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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., (© 2018 British Society for Immunology.)

Background: Recent gains in reducing the global burden of malaria are threatened by the emergence of Plasmodium falciparum resistance to artemisinins. The discovery that mutations in portions of a P. falciparum gene encoding kelch (K13)-propeller domains are the major determinant of resistance has provided opportunities for monitoring such resistance on a global scale., Methods: We analyzed the K13-propeller sequence polymorphism in 14,037 samples collected in 59 countries in which malaria is endemic. Most of the samples (84.5%) were obtained from patients who were treated at sentinel sites used for nationwide surveillance of antimalarial resistance. We evaluated the emergence and dissemination of mutations by haplotyping neighboring loci., Results: We identified 108 nonsynonymous K13 mutations, which showed marked geographic disparity in their frequency and distribution. In Asia, 36.5% of the K13 mutations were distributed within two areas--one in Cambodia, Vietnam, and Laos and the other in western Thailand, Myanmar, and China--with no overlap. In Africa, we observed a broad array of rare nonsynonymous mutations that were not associated with delayed parasite clearance. The gene-edited Dd2 transgenic line with the A578S mutation, which expresses the most frequently observed African allele, was found to be susceptible to artemisinin in vitro on a ring-stage survival assay., Conclusions: No evidence of artemisinin resistance was found outside Southeast Asia and China, where resistance-associated K13 mutations were confined. The common African A578S allele was not associated with clinical or in vitro resistance to artemisinin, and many African mutations appear to be neutral. (Funded by Institut Pasteur Paris and others.).

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.

Background: Cerebral malaria (CM) is one of the major causes of death in African populations infected with Plasmodium falciparum. Only 1% of infected subjects develop CM. The reasons for these differences are not fully understood, but it is likely that the host humoral response against blood-stage antigens plays a role in protection from malaria, although the precise targets and mechanisms mediating immunity remain unclear., Objective: The purpose of this study was to distinguish between defined P. falciparum-specific Ab response patterns in patients presenting with mild malaria (MM) vs. CM., Methods: We used a panel of P. falciparum conserved antigens including crude blood-stage extracts schizont, merozoite and parasitised erythrocyte membranes and MSP-1p19, PfEB200, R23 and GST-5 recombinant antigens in a retrospective case-control study of symptomatic adults, one group presenting confirmed CM without fatal outcome and another group with MM. We further matched P. falciparum-specific Ab responses with those from individuals living in an endemic setting known to have protective immunity and considered them as "immune control" subjects (IC). Total IgG, IgM and IgG subclass Ab responses were determined using ELISA method., Results: Substantial Ab responses were found in symptomatic patients, significantly lower than the "immune control" subjects, and with a limited quantitative difference between MM versus CM. Interestingly, asynchronous IgM response was evidenced in CM contrary to MM., Conclusion: Our results suggest that the contribution of an efficient IgG response against parasite multiplication is of importance in the evolution towards CM manifestation without fatal outcome and deserves further analysis for vaccine candidates.

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.

To investigate the potential for use of a well-established strain of Plasmodium falciparum as a reference strain for infected red blood cell (IRBC) surface reactivity, we monitored the binding of specific immunoglobulin G (IgG) from immune individuals to the reference Knob-positive FCR3 strain by flow cytometry. To permit interassay comparison for 162 plasma samples drawn after the rainy season, a labeling index (LI) was defined as the percentage of labeled parasites multiplied by the mean peak intensity. An LI ratio (LIR) was then calculated as the LI of the sample divided by the LI of the control. LIRs were calculated for individuals living in Dielmo and Ndiop, two Senegalese villages where P. falciparum is transmitted holoendemically and mesoendemically, respectively. The incidence (persons with an LIR of >3) observed in Dielmo was lower than that observed in Ndiop. Significantly higher LIRs were observed (i) for samples from Ndiop than for samples from Dielmo (P < 0.01) and (ii) in Ndiop, in subjects with hemoglobin AS (HbAS) than in those with hemoglobin AA (P = 0.03). No correlation with the cumulative age-associated immune status of the villagers was evidenced, contrary to antibody (Ab) responses against conserved IRBC-associated antigen (Ag) measured by enzyme-linked immunosorbent assay. These results are consistent with the notions that protection in HbAS individuals may relate to an increased IgG response to IRBC membrane Ags and that cell surface reactivity parallels IgG responses even though it is in itself a distinct indicator of the anti-P. falciparum Ab response. Measures of IgG binding to live IRBC are thus relevant for the functional screening of conserved IRBC-associated Ags that contribute to parasite destruction in vivo, as these Ags might be included in a multitarget vaccine.

We conducted an analysis to reevaluate the in vitroculture inhibition assays as a reliable criteria of functional activity of anti-P. falcipanrm antibodies in premunized individuals. Several strains of P. falcipanrm adapted to in vitro culture were compared, and various technical conditions of parasite growth factors such as culture medium or incubation conditions were explored. A subsequent degree of variation was evidenced related to the parasite strain used and the culture conditions. The culture inhibition and the merozoite reinvasion inhibition assays were performed using a collection of plasma from premunized individuals living in two different endemic area of transmission in Senegal. High levels of inhibition were evidenced with a limited degree of variation according to the two different locations of individual's samples. A Significant relationship between anti-merozoite Ab levels and the culture inhibition assays was found contrary to the merozoite re-invasion inhibition assays. Taken together, our results show that such inhibition assays can be managed in facilities of southern laboratories near endemic areas. However, strictly defined culture conditions, homogeneous withdrawals of patients and standardized protocols are necessary for the assessment of such functional assaysas a potential markerof protection-associated mechanisms in longitudinal studies.

To investigate host factors affecting the delay of reappearance of malaria parasites after radical treatment, a study was undertaken in Dielmo, Senegal, an area of intense perennial malaria transmission. A 7-day course of quinine was administered to 173 asymptomatic persons from 1 to 85 years of age and reappearance of malaria parasites in the peripheral blood was monitored weekly for 14 weeks. Additional thick blood films were made in case of fever as part of a daily clinical surveillance. The median times before reappearance of Plasmodium falciparum were 22, 39, and 53 days among persons 1-6, 7-14, and > or = 15 years of age, respectively (P < 0.0001). Multivariate analysis indicated that the daily rate of reappearance of P. falciparum was 2.2 (95% confidence interval [CI] = 1.2-4.5) times lower in sickle cell trait carriers than in AA individuals, and 1.5 (95% CI = 1.1-2.1) times lower in bed nets users than in non-users. The risk ratio for the daily risk of reappearance was significantly related to the level of parasitemia before treatment. No influence of glucose-6-phosphate dehydrogenase deficiency, HLA-B53, and DR13 were observed. Findings show that monitoring during a few weeks the reappearance of malaria parasites after treatment among a small cohort of individuals naturally exposed to malaria is relevant for investigating host resistance factors. This suggest that small, low-cost, field trials may be very informative on the potential of new malaria vaccine candidates.

The B and T cell responses to EB200, a repetitive part of the Plasmodium falciparum antigen Pf332, were examined in malaria-exposed Senegalese adults. Most donors had high levels of antibodies to recombinant EB200 and 17 overlapping peptides spanning EB200. Taking proliferation and/or cytokine (interferon-gamma and interleukin-4) production as a measure of T cell activation, eight of the EB200-derived peptides induced responses in > 40% of the donors tested. There was no general association between the different types of T cell responses measured, emphasizing the importance of including multiple parameters when analyzing T cell responses and suggesting that EB200 induces functionally distinct T cell responses. The most efficient peptide for induction of proliferative responses was one previously shown to induce T cell responses in five different H-2 congenic mouse strains primed with EB200, suggesting that this is a universal T cell epitope. The presence of multiple B and T cell epitopes in EB200, widely recognized by humans, is important since EB200 has been shown to elicit protective antibody responses in monkeys and may be considered for inclusion in malaria subunit vaccines.

This study shows markedly different isotype distributions of antibodies to asexual blood stages of Plasmodium falciparum and to merozoite surface protein 1 in clinically immune Senegalese adults depending on the study site. The relationships between immunoglobulin M (IgM) and IgG and between IgG3 and IgG1 antibodies differed in settings where transmission is perennial compared to settings where it is seasonal. This suggests a role for antibody class and/or subclass production and utilization in the regulation of protective immunity to such antigens.

While the C-terminal repeat region of Pf155/RESA, a Plasmodium falciparum vaccine candidate has been extensively studied for B- and T-cell reactivities, little is so far known about the non-repeat region in this respect. The present study aimed at investigating the non-repeat sequence 171-227 of Pf155/RESA for T- and B-cell epitopes. Eight overlapping peptides were synthesised and assayed for their ability to stimulate peripheral blood mononuclear cells obtained from P. falciparum-immune donors to proliferate and to induce secretion of interferon-gamma (IFN-gamma) and/or interleukin 4 (IL-4) using the ELISPOT assay. The plasmas of the corresponding donors were tested for antibody reactivity with the same peptides in ELISA. The individual cellular responses to the different peptides varied and in general they were not correlated, emphasising the importance of including several parameters for T-cell activation. The most frequent T-cell responses (proliferation, IFN-gamma and/or IL-4) were seen with two partially overlapping peptides corresponding to the sequences 171-185 and 181-195 that induced responses in 71 and 62% of the donors, respectively. Although, the frequency of responders was high, the magnitude of the responses was generally low. Two overlapping peptides corresponding to the sequence 186-206 bound antibodies from a large number of plasma samples. IL-4 producing cells were frequently found in donors whose sera contained antibodies to the corresponding peptide. However, there was no absolute correlation and many donors having anti-peptide antibodies could also be induced to produce IFN-gamma. In conclusion, the non-repeat region of Pf155/RESA contains several epitopes inducing functionally distinct T-cell responses. The sequence 171-206 was found to contain both B- and T-cell epitopes recognised by almost all individuals naturally primed to malaria. Thus, this sequence should be a useful tool in future immuno-epidemiological studies and/or for inclusion into a subunit vaccine against the asexual blood stages of the P. falciparum parasite.

Background: The sickle-cell trait protects against severe Plasmodium falciparum malaria and reduces susceptibility to mild malaria but does not prevent infection. The exact mechanism of this protection remains unclear. We have hypothesized that AS individuals are protected by virtue of being less susceptible to a subset of parasite strains; thus we compared some genetic characteristics of parasites infecting AS and AA subjects., Materials and Methods: Blood was collected from asymptomatic individuals living in two different regions of Africa. The polymorphic MSP-1 and MSP-2 loci were genotyped using a PCR-based methodology. Individual alleles were identified by size polymorphism, amplification using family-specific primers, and hybridization using family-specific probes. Multivariate logistic regression was used to analyze allele distribution., Results: In Senegalese carriers, age and hemoglobin type influenced differently the distribution of the three MSP-1 families and had an impact on distinct individual alleles, whereas the distribution of MSP-2 alleles was marginally affected. There was no influence of other genetic traits, including the HLA Bw53 genotype, or factors such as place of residence within the village. In a cohort of Gabonese schoolchildren in which the influence of age was abrogated, a similar imbalance in the MSP-1 allelic distribution but not of MSP-2 allelic distribution by hemoglobin type was observed., Conclusions: The influence of the host's hemoglobin type on P. falciparum genotypes suggests that parasite fitness for a specific host is strain-dependent, which is consistent with our hypothesis that innate resistance might result from reduced fitness of some parasite strains for individuals with sickle-cell traits.

Forty-one African patients suffering from clinically defined severe malaria were studied in the intensive medical care unit of the main hospital in Dakar, Senegal, West Africa. All of these individuals lived in Greater Dakar, an area of low and seasonal Plasmodium falciparum endemicity. Twenty-seven patients (mean age +/- 1 standard deviation, 19.2 +/- 12.7 years) survived this life-threatening episode, but 14 (30.8 +/- 16.2 years old) died despite initiation of adequate treatment. On the day of admission (day 0) and 3 days later, one to two blood samples (i.e., approximately 10 to 15 ml) were obtained from each subject, and different biological parameters were evaluated in the two groups. Plasma samples were tested for their content in tumor necrosis factor alpha (TNF-alpha), soluble receptors I and II for TNF-alpha (TNF-alpha sRI and TNF-alpha sRII), interleukin-6 (IL-6), IL-6 sR, IL-10, and IL-2 sR. The concentrations of all these cytokines and/or their receptors was significantly elevated in patient plasma samples on day 0, and it rapidly decreased in the group of individuals who survived. By comparison, the mean concentration of the same parameters decreased slowly in the group of patients who died (except for IL-10, which dramatically fell in all patient plasma samples soon after initiation of antimalarial treatment). The TNF-alpha sRI level remained significantly elevated among the patients who died, and the highest levels of soluble TNF-alpha sRI receptor were found among the older patients. Parasite-specific immunoglobulin M (IgM), total IgG, IgG1, IgG2, IgG3, and IgG4 were evaluated by enzyme-linked immunosorbent assay using a crude extract of a local P. falciparum isolate as antigen and human class- and subclass-specific monoclonal antibodies. Parasite-specific IgM, total IgG, and IgG1 were detectable in the plasma samples of most of these African patients, whereas IgG2 and IgG4 mean values were low. The mean level of parasite-specific IgG3 was different (P = 0.024) at day 0, i.e., before initiation of intensive medical care, between the group of the 27 surviving subjects and the group of 14 patients dying of severe malaria. As a consequence, most of the African patients who died had only trace amounts or almost no detectable level of parasite-specific IgG3 at the time of admission. In contrast, the presence of even limited IgG3 activity at day 0 was found to be associated with a significantly increased probability of recovering from severe malaria. Therefore, in our study, both an elevated level of TNF-alpha sRI and absence of IgG3 activity were of bleak prognostic significance, whereas a favorable outcome was usually observed when parasite-specific IgG3 activity was detectable. This finding was strongly suggestive of a prime role for these parasite-specific immunoglobulins in the capacity to help recovery from severe malaria.

Specific immune responses to asexual blood stages of P. falciparum antigens (a lysate of parasitized red blood cells and a characterized vaccine candidate i.e. MSP1 p19) were analyzed in plasma samples from immune adult individuals living in three different areas of Senegal, where malaria transmission is different. Most individuals in the three sites had specific IgG and IgM to total P. falciparum antigens, whereas approximately 50% had either IgG or IgM specific to MSP1 p19. Further, no anti-MSP1 p19 IgG2 and IgG4 antibody was noticed in any individual whereas the distribution of anti-MSP1 p19 IgG1 and IgG3 was different upon the epidemiological context. In addition, no relationship was found between antibody responses and in vitro T cell responses against P. falciparum antigens upon those experimental conditions. These data stress on the relatively elevated distribution of specific antibodies to MSP1 p19 in P. falciparum hyperendemic areas and suggest a differential regulation of isotypes depending on individual parasite exposure.

Crude merozoite antigens from P. falciparum were used to analyse the proliferative response of peripheral blood mononuclear cells from 114 inhabitants of the village of Dielmo (Senegal, West Africa), who are exposed continuously to malaria transmission. The high or low responses to merozoite antigens obtained in lymphocyte stimulation assays were correlated to the presence or absence of parasites, to the IFN-gamma production and to the HLA-phenotype. High responders produced high levels of IFN-gamma while low responders did not secrete IFN-gamma (23/27). The two HLA phenotypes HLA-B51 and HLA-DR1 were significantly associated with high response (p < 0.05).

In the aim to determine the possible role of HLA-antigens in malaria infection, sera from 50 HLA-typed donors from Dielmo (Senegal) were tested in immunoblotting (using crude merozoites as antigen) and immunoprecipitation (using detergent-extracts from surface-iodinated merozoite as antigen). The donors were previously tested on lymphocyte proliferation in vitro and gamma-interferon production and grouped into two classes: high responders and low responders. In immunoblotting and immunoprecipitation experiments, no specific differences were found in the antibody reactivity with native merozoite antigen in individuals with high (HR) or low (LR) in vitro proliferative T cell responses. In other words, both groups of responders, high and low, showed antibodies in their sera against a wide range of different parasite antigens; although between individual donors striking differences were found. Individual donors had developed different levels of antibodies, or no antibodies at all, against individual natural antigens. These differences, however, could not be correlated with HR or LR. The band patterns obtained were compared with HLA-antigens of donors phenotypes. Results showed that there was no correlation found between the different merozoite antigens recognized by sera of the different donors or groups of donors (HR and LR) and the donors' HLA-phenotypes. The fact that donors with HLA-B51 all recognized (MSP1(42) and donors with DR1 recognized MSP1(19), was not a convincing correlation.

Pf72/Hsp70-1, a heat-shock protein of m.w. 72 kDa from Plasmodium falciparum is one of the Ag of interest to be included in a polyvalent vaccine against malaria. It is one of the major immunogens present in a fraction of purified blood stage parasites that elicited protection against experimental infection of Saimiri monkeys with blood stages of P. falciparum. It is present at all blood stages and one of its B cell epitopes is also detected on the surface of the infected hepatocyte. Moreover, Pf72 appears to be well conserved among different isolates of P. falciparum. We have examined the immune response against Pf72/Hsp70-1 in individuals from different age groups living in a holoendemic area (West Africa). The immune response against the native Ag (purified from schizonts and called Pf/Hsp70) was analyzed both at the humoral level by ELISA and at the cellular level by assessing in vitro proliferation and IFN-gamma production of PBMC. Of the individuals studied 52% had a statistically significant level of anti-Pf/Hsp70 antibodies as compared with unexposed individuals. These positive individuals showed a heterogeneous distribution because significant levels of antibodies were found in 70% of the adults but in only 26% of the children. The presence of Pf/Hsp70-specific reactive T cells in the blood was detected in 32% of the individuals. The total anti-Pf/Hsp70 antibody level (IgG+IgM) appeared strongly age related and correlated positively with parasite exposure, whereas the T cell response failed to correlate either with the antibody level or with age. Moreover, PBMC of donors responded to the Pf/Hsp70 in a dissociated way, namely, by either T cell proliferation or IFN-gamma production. Ten synthetic peptides based on sequences found in the C-terminal part of Pf72/Hsp70-1 were further tested as potential T cell epitopes. The proliferative response of PBMC from individuals continuously exposed to the parasite showed that three peptides more frequently trigger significant T cell proliferation (in 21% to 27% of the individuals) and three others less frequently (10%). None of these peptides allowed detection of reactive T cells in PBMC of Europeans with no previous exposure to malaria. Some of the stimulating peptides are highly similar to human heat-shock Hsc and Hsp70 with large stretches of identical amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)

T CELL response in vitro to merozoite extracts of P falciparum was tested in holoendémic area (Dielmo, Region of Fatick). 58 individuals (29 adults and 29 children) were sampled. Mononuclear cells from peripheral blood were cultured with the presence of merozoites antigens during 7 days. Cells were pulsed at day 6 and 3H-thymidine incorporation was measured 16h later. T Cell response high or low was evaluated by Stimulation Index (SI). This is defined as mean cpm expérimental cells/mean cpm control cells. Two groups of Responders have been identified: High Résponders: SI > or = 5 48% of subjects Low Responders: SI < 5 52% of subjects. A microparasitaemia have been found in 19 donnors without illness. 14 of these are Low Responders (89%). It was found that SI increased with the number year of exposure.

Sequestration is a phenomenon where mature Plasmodium falciparum infected-erythrocytes block microvessels. Many cells (particularly epithelial cells) can cytoadhere to these erythrocytes and this may play an important role in the physiopathology of cerebral malaria. Many in vitro models have been proposed. They permitted different studies about cytoadherence and clinical effects. Contradictory results have been yet published. Investigations at a molecular level of cytoadherence moiety contribute to a better understanding of physiopathology and therapeutic improvements of cerebral malaria.

Malaria is an important public health problem in developing countries. The production of a good vaccine needs a better knowledge of different mechanisms which control the immune system. Results from donors show a higher frequency of antigens HLA: A2, B35, CW4, CW6, DRW52, DQW1 and DQW3. The lymphoproliferation in vitro of mononuclear cells, T cells and CD4+ and CD8+ subsets T cells with merozoite extracts is not so clear to establish a correlation between HLA phenotypes and T cells response.

Abstract Malaria infection is a multifactorial disease partly modulated by host immuno-genetic factors. Recent evidence has demonstrated the importance of Interleukin-17 family proinflammatory cytokines and their genetic variants in host immunity. However, limited knowledge exists about their role in parasitic infections such as malaria. We aimed to investigate IL-17A serum levels in patients with severe and uncomplicated malaria and gene polymorphism’s influence on the IL-17A serum levels. In this research, 125 severe (SM) and uncomplicated (UM) malaria patients and 48 free malaria controls were enrolled. IL-17A serum levels were measured with ELISA. PCR and DNA sequencing were used to assess host genetic polymorphisms in IL-17A. We performed a multivariate regression to estimate the impact of human IL-17A variants on IL-17A serum levels and malaria outcomes. Elevated serum IL-17A levels accompanied by increased parasitemia were found in SM patients compared to UM and controls (P

Abstract Background Genome-wide association studies have identified ATP2B4 as a severe malaria resistance gene. Recently, 8 potential causal regulatory variants have been shown to be associated with severe malaria. Methods Genotyping of rs10900585, rs11240734, rs1541252, rs1541253, rs1541254, rs1541255, rs10751450, rs10751451 and rs10751452 was performed in 154 unrelated individuals (79 controls and 75 mild malaria patients). rs10751450, rs10751451 and rs10751452 were genotyped by Taqman assays, whereas the fragment of the ATP2B4 gene containing the remaining SNPs was sequenced. Logistic regression analysis was used to assess the association between the SNPs and mild malaria. Results The results showed that mild malaria was associated with rs10900585, rs11240734, rs1541252, rs1541253, rs1541254, rs1541255, rs10751450, rs10751451 and rs10751452. The homozygous genotypes for the major alleles were associated with an increased risk of mild malaria. Furthermore, the haplotype containing the major alleles and that containing the minor alleles were the most frequent haplotypes. Individuals with the major haplotypes had a significantly higher risk of mild malaria compared to the carriers of the minor allele haplotype. Conclusions ATP2B4 polymorphisms that have been associated with severe malaria are also associated with mild malaria.

Abstract Background The diagnosis of malaria cases in regions where the malaria burden has decreased significantly and prevalence is very low is more challenging, in part because of reduced clinical presumption of malaria. The appearance of a cluster of malaria cases with atypical symptoms in Mbounguiel, a village in northern Senegal where malaria transmission is low, in September 2018 exemplifies this scenario. The collaboration between the National Malaria Control Programme (NMCP) at the Senegal Ministry of Health and the Laboratory of Parasitology and Mycology at Cheikh Anta Diop University worked together to evaluate this cluster of malaria cases using molecular and serological tools. Methods Malaria cases were diagnosed primarily by rapid diagnostic test (RDT), and confirmed by photo-induced electron transfer-polymerase chain reaction (PET-PCR). 24 single nucleotide polymorphisms (SNPs) barcoding was used for Plasmodium falciparum genotyping. Unbiased metagenomic sequencing and Luminex-based multi-pathogen antibody and antigen profiling were used to assess exposure to other pathogens. Results Nine patients, of 15 suspected cases, were evaluated, and all nine samples were found to be positive for P. falciparum only. The 24 SNPs molecular barcode showed the predominance of polygenomic infections, with identifiable strains being different from one another. All patients tested positive for the P. falciparum antigens. No other pathogenic infection was detected by either the serological panel or metagenomic sequencing. Conclusions This work, undertaken locally within Senegal as a collaboration between the NMCP and a research laboratory at University of Cheikh Anta Diop (UCAD) revealed that a cluster of malaria cases were caused by different strains of P. falciparum. The public health response in real time demonstrates the value of local molecular and genomics capacity in affected countries for disease control and elimination.

Abstract Background Molecular epidemiology can provide important information regarding the genetic diversity and transmission of Plasmodium falciparum, which can assist in designing and monitoring elimination efforts. However, malaria molecular epidemiology including understanding the genetic diversity of the parasite and performing molecular surveillance of transmission has been poorly documented in Senegal. Next Generation Sequencing (NGS) offers a practical, fast and high-throughput approach to understand malaria population genetics. This study aims to unravel the population structure of P. falciparum and to estimate the allelic diversity, multiplicity of infection (MOI), and evolutionary patterns of the malaria parasite using the NGS platform. Methods Multiplex amplicon deep sequencing of merozoite surface protein 1 (PfMSP1) and merozoite surface protein 2 (PfMSP2) in fifty-three P. falciparum isolates from two epidemiologically different areas in the South and North of Senegal, was carried out. Results A total of 76 Pfmsp1 and 116 Pfmsp2 clones were identified and 135 different alleles were found, 56 and 79 belonged to the pfmsp1 and pfmsp2 genes, respectively. K1 and IC3D7 allelic families were most predominant in both sites. The local haplotype diversity (Hd) and nucleotide diversity (π) were higher in the South than in the North for both genes. For pfmsp1, a high positive Tajima’s D (TD) value was observed in the South (D = 2.0453) while negative TD value was recorded in the North (D = − 1.46045) and F-Statistic (Fst) was 0.19505. For pfmsp2, non-directional selection was found with a highly positive TD test in both areas and Fst was 0.02111. The mean MOI for both genes was 3.07 and 1.76 for the South and the North, respectively, with a statistically significant difference between areas (p = 0.001). Conclusion This study revealed a high genetic diversity of pfmsp1 and pfmsp2 genes and low genetic differentiation in P. falciparum population in Senegal. The MOI means were significantly different between the Southern and Northern areas. Findings also showed that multiplexed amplicon deep sequencing is a useful technique to investigate genetic diversity and molecular epidemiology of P. falciparum infections.

Abstract Background Population-wide interventions using malaria testing and treatment might decrease the reservoir of Plasmodium falciparum infection and accelerate towards elimination. Questions remain about their effectiveness and evidence from different transmission settings is needed. Methods A pilot quasi-experimental study to evaluate a package of population-wide test and treat interventions was conducted in six health facility catchment areas (HFCA) in the districts of Kanel, Linguère, and Ranérou (Senegal). Seven adjacent HFCAs were selected as comparison. Villages within the intervention HFCAs were stratified according to the 2013 incidences of passively detected malaria cases, and those with an incidence ≥ 15 cases/1000/year were targeted for a mass test and treat (MTAT) in September 2014. All households were visited, all consenting individuals were tested with a rapid diagnostic test (RDT), and, if positive, treated with dihydroartemisinin-piperaquine. This was followed by weekly screening, testing and treatment of fever cases (PECADOM++) until the end of the transmission season in January 2015. Villages with lower incidence received only PECADOM++ or case investigation. To evaluate the impact of the interventions over that transmission season, the incidence of passively detected, RDT-confirmed malaria cases was compared between the intervention and comparison groups with a difference-in-difference analysis using negative binomial regression with random effects on HFCA. Results During MTAT, 89% (2225/2503) of households were visited and 86% (18,992/22,170) of individuals were tested, for a combined 77% effective coverage. Among those tested, 291 (1.5%) were RDT positive (range 0–10.8 by village), of whom 82% were

Abstract Background Malaria surveillance requires powerful tools and strategies to achieve malaria elimination. Rapid diagnostic tests for malaria (RDTs) are easily deployed on a large scale and are helpful sources of parasite DNA. The application of sensitive molecular techniques to these RDTs is a modern tool for improving malaria case detection and drug resistance surveillance. Several studies have made it possible to extract the DNA of Plasmodium falciparum from RDTs. The knowledge of gametocyte carriage in the population is important to better assess the level of parasite transmission in elimination settings. The aim of this study was to detect P. falciparum gametocytes from used RDTs by quantitative PCR for molecular monitoring of malaria transmission. Methods DNA was extracted from 303 RDT devices (SD Bioline Malaria Pf) using the Chelex-100 protocol. qPCR was performed in a 20 μL reaction to detect and quantify transcripts of the pfs25 gene. The cycle threshold (Ct) was determined by the emission fluorescence corresponding to the initial amount of amplified DNA. Results The study found an overall prevalence of 53.47% with an average Ct of 32.12 ± 4.28 cycles. In 2018, the prevalence of gametocytes was higher in the Ranérou district (76.24%) than in the Saint-Louis district (67.33%) where an increase in the number of gametocyte carriers in 2018 was noted, in comparison with 2017. Conclusions RDTs are a good source of DNA for molecular monitoring of gametocyte carriage. This method is a simple and effective tool to better understand the level of malaria transmission with a view to elimination.

Abstract Background Severe forms of malaria (SM) are an outcome of Plasmodium falciparum infection and can cause death especially in children under 4 years of age. RNASE3 (ECP) has been identified as an inhibitor of Plasmodium parasites growth in vitro, and genetic analysis in hospitalized Ghanaian subjects has revealed the RNASE3 +371G/C (rs2073342) polymorphism as a susceptibility factor for cerebral malaria. The +371 C allele results in an Arg/Thr mutation that abolishes the cytotoxic activity of the ECP protein. The present study aims to investigate RNASE3 gene polymorphisms and their putative link to severe malaria in a malaria cohort from Senegal. Methods/results Patients enrolled from hospitals were classified as having either uncomplicated (UM) or severe malaria (SM). The analysis of the RNASE3 gene polymorphisms was performed in 241 subjects: 178 falciparum infected (96 SM, 82 UM) and 63 non-infected subjects as population control group (CTR). Six frequent SNPs (MAF > 3%) were identified, and one SNP was associated with malaria severity by performing a logistic regression analysis SM vs.UM: RNASE3 +499G/C (rs2233860) under age, sex as covariates and HbS/HbC polymorphisms adjustment (p = 0.003, OR 0.43, CI 95% 0.20–0.92). The polymorphisms: +371G/C (rs2073342), +499G/C (rs2233860) and +577A/T (rs8019343) defined a haplotype risk (G-G-T) for malaria severity (Fisher exact test, p = 0.03) (OR 4.1, IC 95% (1.1–14.9). Conclusion In addition to the previously described association of +371G/C polymorphism in Ghanaians cohort, the RNASE3 +499G/C polymorphism was associated with susceptibility to SM in a Senegalese population. The haplotype +371G/+499G/+577T defined by RNASE3 polymorphisms was associated with severity. The genetic association identified independently in the Senegalese population provide additional evidence of a role of RNASE3 (ECP) in malaria severity.

Abstract Background Malaria is a leading cause of mortality and morbidity in tropical countries, especially in sub-Saharan Africa. In Senegal, a control plan implemented in the beginning of the 2000s has enabled a substantial reduction of mortality and morbidity due to malaria. However, eradication of malaria requires a vaccine that protects against Plasmodium falciparum the deadliest species of the parasite that causes this disease. Plasmodium falciparum is characterized by an extensive genetic diversity that makes vaccine development challenging. In this study, the diversity of P. falciparum isolates was analysed from asymptomatic children residing in the district of Toubacouta, Senegal. Methods A nested PCR approach was used to perform genotyping of the msp-1 and msp-2 loci in samples from 87 asymptomatic children infected with P. falciparum, collected during a cross sectional survey in November and December 2010. Parasite densities in blood samples were determined by microscopic examination and statistical analyses were used to identify association of parasite genotype and parasitaemia. Results Genotyping was successful in 84/87 and 82/87 samples for msp-1 and msp-2, respectively. A strong genetic diversity was found with a total of 15 and 21 different alleles identified for msp-1 and msp-2, respectively. RO33 was the most frequent allelic family of msp-1 followed by MAD20, then by K1. Regarding msp-2 allelic families, 3D7 was more common than FC27. Multiple infections were predominant, since 69% and 89% of the samples genotyped for msp-1 and msp-2 showed more than one clone of P. falciparum with complexity of infection (COI) of 2.5 and 4.7, respectively. Expected heterozygosity (HE) was 0.57 and 0.55 for msp-1 and msp-2, respectively. Interestingly, polyclonal infections were significantly associated with higher parasitaemia. Conclusions The strong genetic diversity of P. falciparum clones and the association of polyclonal infection with high parasitaemia call for a multi-allelic approach in the design of vaccine candidates for efficient malaria eradication.

Abstract Background The monitoring of Plasmodium falciparum sensitivity to anti-malarial drugs is a necessity for effective case management of malaria. This species is characterized by a strong resistance to anti-malarial drugs. In Senegal, the first cases of chloroquine resistance were reported in the Dakar region in 1988 with nearly 7% population prevalence, reaching 47% by 1990. It is in this context that sulfadoxine–pyrimethamine temporarily replaced chloroquine as first line treatment in 2003, pending the introduction of artemisinin-based combination therapy in 2006. The purpose of this study is to assess the ex vivo sensitivity to different anti-malarial drugs of the P. falciparum population from Pikine. Methods Fifty-four samples were collected from patients with non-complicated malaria and aged between 2 and 20 years in the Deggo health centre in Pikine in 2014. An assay in which parasites are stained with 4′, 6-di-amidino-2-phenylindole (DAPI), was used to study the ex vivo sensitivity of isolates to chloroquine, amodiaquine, piperaquine, pyrimethamine, and dihydroartemisinin. High resolution melting was used for genotyping of pfdhps, pfdhfr, pfmdr1, and pfcrt genes. Results The mean IC50s of chloroquine, amodiaquine, piperaquine, dihydroartemisinin, and pyrimethamine were, respectively, 39.44, 54.02, 15.28, 2.23, and 64.70 nM. Resistance mutations in pfdhfr gene, in codon 437 of pfdhps gene, and an absence of mutation at position 540 of pfdhps were observed. Mutations in codons K76T of pfcrt and N86Y of pfmdr1 were observed at 51 and 11% population prevalence, respectively. A relationship was found between the K76T and N86Y mutations and ex vivo resistance to chloroquine. Conclusion An increase in sensitivity of isolates to chloroquine was observed. A high sensitivity to dihydroartemisinin was observed; whereas, a decrease in sensitivity to pyrimethamine was observed in the parasite population from Pikine.

Abstract Background Emergence and spread of drug resistance to every anti-malarial used to date, creates an urgent need for development of sensitive, specific and field-deployable molecular tools for detection and surveillance of validated drug resistance markers. Such tools would allow early detection of mutations in resistance loci. The aim of this study was to compare common population signatures and drug resistance marker frequencies between two populations with different levels of malaria endemicity and history of anti-malarial drug use: Tanzania and Sénégal. This was accomplished by implementing a high resolution melting assay to study molecular markers of drug resistance as compared to polymerase chain reaction–restriction fragment length polymorphism (PCR/RFLP) methodology. Methods Fifty blood samples were collected each from a lowly malaria endemic site (Sénégal), and a highly malaria endemic site (Tanzania) from patients presenting with uncomplicated Plasmodium falciparum malaria at clinic. Data representing the DHFR were derived using both PCR–RFLP and HRM assay; while genotyping data representing the DHPS were evaluated in Senegal and Tanzania using HRM. Msp genotyping analysis was used to characterize the multiplicity of infection in both countries. Results A high prevalence of samples harbouring mutant DHFR alleles was observed in both population using both genotyping techniques. HRM was better able to detect mixed alleles compared to PCR/RFLP for DHFR codon 51 in Tanzania; and only HRM was able to detect mixed infections from Senegal. A high prevalence of mutant alleles in DHFR (codons 51, 59, 108) and DHPS (codon 437) were found among samples from Sénégal while no mutations were observed at DHPS codons 540 and 581, from both countries. Overall, the frequency of samples harbouring either a single DHFR mutation (S108N) or double mutation in DHFR (C59R/S108N) was greater in Sénégal compared to Tanzania. Conclusion Here the results demonstrate that HRM is a rapid, sensitive, and field-deployable alternative technique to PCR–RFLP genotyping that is useful in populations harbouring more than one parasite genome (polygenomic infections). In this study, a high levels of resistance polymorphisms was observed in both dhfr and dhps, among samples from Tanzania and Sénégal. A routine monitoring by molecular markers can be a way to detect emergence of resistance involving a change in the treatment policy.

We here analyze data from the first year of an ongoing nationwide program of genetic surveillance of Plasmodium falciparum parasites in Senegal. The analysis is based on 1097 samples collected at health facilities during passive malaria case detection in 2019; it provides a baseline for analyzing parasite genetic metrics as they vary over time and geographic space. The study's goal was to identify genetic metrics that were informative about transmission intensity and other aspects of transmission dynamics, focusing on measures of genetic relatedness between parasites. We found the best genetic proxy for local malaria incidence to be the proportion of polygenomic infections (those with multiple genetically distinct parasites), although this relationship broke down at low incidence. The proportion of related parasites was less correlated with incidence while local genetic diversity was uninformative. The type of relatedness could discriminate local transmission patterns: two nearby areas had similarly high fractions of relatives, but one was dominated by clones and the other by outcrossed relatives. Throughout Senegal, 58% of related parasites belonged to a single network of relatives, within which parasites were enriched for shared haplotypes at known and suspected drug resistance loci and at one novel locus, reflective of ongoing selection pressure. Senegal has initiated a national sentinel surveillance program for malaria parasite genetics. Here, the authors report data from the first year of the program and use it to investigate local malaria incidence, patterns of transmission, and genetic loci under selection. [ABSTRACT FROM AUTHOR]

Background Host factors, including host genetic variation, have been shown to influence the outcome of Plasmodium falciparum infection. Genome-wide linkage studies have mapped mild malaria resistance genes on chromosome 6p21, whereas NCR3-412 polymorphism (rs2736191) lying within this region was found to be associated with mild malaria. Methods Blood samples were taken from 188 Plasmodium falciparum malaria patients (76 mild malaria patients, 85 cerebral malaria patients, and 27 severe non-cerebral malaria patients). NCR3-412 (rs2736191) was analysed by sequencing, and haematological parameters were measured. Finally, their association with clinical phenotypes was assessed. Results We evidenced an association of thrombocytopenia with both cerebral malaria and severe non-cerebral malaria, and of an association of high leukocyte count with cerebral malaria. Additionally, we found no association of NCR3-412 with either cerebral malaria, severe non-cerebral malaria, or severe malaria after grouping cerebral malaria and severe non-cerebral malaria patients. Conclusions Our results suggest that NCR3 genetic variation has no effect, or only a small effect on the occurrence of severe malaria, although it has been strongly associated with mild malaria. We discuss the biological meaning of these results. Besides, we confirmed the association of thrombocytopenia and high leukocyte count with severe malaria phenotypes.

Background. With 214 million cases and 438,000 deaths in 2015, malaria remains one of the deadliest infectious diseases in tropical countries. Several species of the protozoan Plasmodium cause malaria. However, almost all the fatalities are due to Plasmodium falciparum, a species responsible for the severest cases including cerebral malaria. Immune response to Plasmodium falciparum infection is mediated by the production of pro-inflammatory cytokines, chemokines and growth factors whose actions are crucial for the control of the parasites. Following this response, the induction of anti-inflammatory immune mediators downregulates the inflammation thus preventing its adverse effects such as damages to various organs and death. Methods. We performed a retrospective, nonprobability sampling study using clinical data and sera samples from patients, mainly adults, suffering of non-cerebral or cerebral malaria in Dakar, Sénégal. Healthy individuals residing in the same area were included as controls. We measured the serum levels of 29 biomarkers including growth factors, chemokines, inflammatory and anti-inflammatory cytokines. Results. We found an induction of both pro- and anti-inflammatory immune mediators during malaria. The levels of pro-inflammatory biomarkers were higher in the cerebral malaria than in the non-cerebral malaria patients. In contrast, the concentrations of anti-inflammatory cytokines were comparable in these two groups or lower in CM patients. Additionally, four pro-inflammatory biomarkers were significantly increased in the deceased of cerebral malaria compared to the survivors. Regarding organ damage, kidney failure was significantly associated with death in adults suffering of cerebral malaria. Conclusions. Our results suggest that a poorly controlled inflammatory response determines a bad outcome in African adults suffering of cerebral malaria.