Red blood cell polymorphisms in relation to Plasmodium falciparum asymptomatic parasite densities and morbidity in Senegal

Numerous studies have shown that several red blood cell polymorphisms protect against severe malaria. Such a relation is much less clear for mild malaria attacks and for the asymptomatic carriage of Plasmodium falciparum. The impact of red blood cell polymorphisms on the level of parasite density was assessed in a group of 464 Senegalese children from the Sereer ethnic group, studied for 18 months. These genetic factors were also related to the malarial morbidity, investigated during 2 successive transmission seasons among 169 of these children. The frequencies of the host genetic factors in the whole group were 0.52 for blood group O, 0.13 for hemoglobin S, 0.16 for the G6PD A-deficient variant and 0.24 for alpha+-thalassemia (-alpha(3.7) deletion). Hemoglobin S was associated with protection against mild malaria attacks. None of the genetic factors was implicated in a better control of parasite densities. These associations may be particular to this ethnic group due to the specificities of malaria endemicity in this area. The pressure exerted in the area by other non-malarial infectious diseases as well as the genetic heterogeneity of circulating parasites may also contribute to these observations.     

Plasmodium falciparum clinical malaria: lessons from longitudinal studies in Senegal

Development of new antimalaria strategies and particularly vaccines, needs an in-depth understanding of the relationships between transmission, infection, immunity, morbidity and mortality. The intensive and longitudinal collection of entomological, parasitological and clinical data from the Senegalese populations of Dielmo (250-300 inhabitants), exposed to a perennial and intense transmission (about 200 infective bites/person/year) and of Ndiop (300-350 inhabitants) exposed to a seasonal transmission (about 20 infective bites/person/year), allows to respond to many questions about this subject. The acquisition of an antimalaria immunity as one gets older appears to reduce parasite density, complexity of infection, risk of new patent infection after a suppressive treatment but does not reduce the prevalence (as assessed by PCR) of infection which is commonly chronic and asymptomatic. The existence of a pyrogenic threshold effect of parasitaemia allows the individual diagnosis of malaria attacks. P. falciparum genotyping suggests that successive malaria attacks are due to distinct recently inoculated parasite populations that multiply initially without restriction, a dominant population is generally responsible of the clinical manifestations and all new populations do not trigger systematically attacks. The initial intensity of clinical manifestations does not differ perceptibly among children and adults, is not related to the duration of the attacks, does not allow the distinction between several types of attacks, is not predictive of their severity, and the clearance of parasites and manifestations is longer among youngest persons. The risk of malaria attacks is lower as one gets older and among carriers of AS haemoglobin, is higher when transmission increases and during pregnancy up to three months after delivery, and vary between children. The risk of malaria attack per infective bite is negatively related to the intensity of transmission. Because of their high sensitivity in malaria case detection, this type of small community-based studies are powerful and useful for the identification of protective immunological mechanisms as well as for testing rapidly and cheaply the clinical efficacy of any intervention such as antimalarial vaccines and drug therapy or prophylaxis. As a lot of vaccine candidates and drug combinations will be screened or tested in the perspective of the 'Roll-Back Malaria' programme, more attention must be given to longitudinal studies of this type.     

Transmission dynamics of Plasmodium falciparum

Recent models of malaria have been developed by Gupta and her co-workers. A frequent assumption used to illustrate these models is that levels of malaria are controlled by lifelong strain-specific immunity. In this article, Allan Saul examines the predictions this model makes about the equilibrium values of parasite prevalence and the dynamics of an epidemic following the introduction of a new strain. He reaches the conclusion that the stability of malaria makes long-term strain-specific immunity highly improbable, thus rendering models requiring lifelong strain-specific immunity unlikely to be of relevance in most epidemiological contexts.     

Genetic diversity and population structure of Plasmodium falciparum isolates from Dakar,Senegal, investigated from microsatellite and antigen determinant loci

We investigated the genetic diversity and the population structure of 32 Plasmodium falciparum blood sample isolates (25 from Dakar city and suburbs and seven from other localities in Senegal) with two different types of molecular markers, 19 microsatellite and four antigenic determinant loci. Under the same technical procedure, microsatellite loci showed a mean number of alleles greater than that of antigenic loci. Both markers revealed that 15.6% of blood samples were multi-infected. Mean expected heterozygosity calculated from microsatellites and antigens was similar, 0.74 and 0.70, respectively. Significant linkage disequilibrium was observed from microsatellite loci and antigenic determinant loci. This suggests a non-panmictic structure for this sample that could be explained by two non-exclusive hypotheses: (i) a particular mating system (i.e. clonality), and/or (ii) a population structure in P. falciparum (i.e. Wahlund effect). Urban samples could have been drawn from a heterogeneous set of foci with different level of parasitic transmission. Moreover, no relationship was found between multilocus genotypes and different parameters (i.e. age, sex and blood group of parasitized patients; number of trophozoites per microliter of blood). The results are discussed taking into account recently published studies on malaria population biology.     

Multiclonal asymptomatic Plasmodium falciparum infections predict a reduced risk of malaria disease in a Tanzanian population

Protective immunity to malaria is acquired after repeated exposure to the polymorphic Plasmodium falciparum parasite. Whether the number of concurrent antigenically diverse clones in asymptomatic infections predicts the risk of subsequent clinical malaria needs further understanding. We assessed the diversity of P. falciparum infections by merozoite surface protein 2 genotyping in a longitudinal population based study in Tanzania. The number of clones was highest in children 6-10 years and in individuals with long time to previous anti-malarial treatment. Individual exposure, analysed by circumsporozoite protein antibody levels, was associated with parasite prevalence but not with the number of clones. The risk of subsequent clinical malaria in children free of acute disease or recent treatment was, compared to one clone, reduced in individuals with multiclonal infections or without detectable parasites, with the lowest hazard ratio 0.28 (95% confidence interval 0.10-0.78 Cox regression) for 2-3 clones. The number of clones was not associated with haemoglobin levels. A reduced risk of malaria in asymptomatic individuals with multiclonal persistent P. falciparum infections suggests that controlled maintenance of diverse infections is important for clinical protection in continuously exposed individuals, and needs to be considered in the design and evaluation of new malaria control strategies.     

An integrated model of Plasmodium falciparum dynamics

The within-host and between-host dynamics of malaria are linked in myriad ways, but most obviously by gametocytes, the parasite blood forms transmissible from human to mosquito. Gametocyte dynamics depend on those of non-transmissible blood forms, which stimulate immune responses, impeding transmission as well as within-host parasite densities. These dynamics can, in turn, influence antigenic diversity and recombination between genetically distinct parasites. Here, we embed a differential-equation model of parasite-immune system interactions within each of the individual humans represented in a discrete-event model of Plasmodium falciparum transmission, and examine the effects of human population turnover, parasite antigenic diversity, recombination, and gametocyte production on the dynamics of malaria. Our results indicate that the local persistence of P. falciparum increases with turnover in the human population and antigenic diversity in the parasite, particularly in combination, and that antigenic diversity arising from meiotic recombination in the parasite has complex differential effects on the persistence of founder and progeny genotypes. We also find that reductions in the duration of individual human infectivity to mosquitoes, even if universal, produce population-level effects only if near-absolute, and that, in competition, the persistence and prevalence of parasite genotypes with gametocyte production concordant with data exceed those of genotypes with higher gametocyte production. This new, integrated approach provides a framework for investigating relationships between pathogen dynamics within an individual host and pathogen dynamics within interacting host and vector populations.     

Prevalence of molecular markers of Plasmodium falciparum drug resistance in Dakar, Senegal

ABSTRACT: BACKGROUND: As a result of the widespread resistance to chloroquine and sulphadoxinepyrimethamine, artemisinin-based combination therapy (ACT) (including artemetherlumefantrine and artesunate-amodiaquine) has been recommended as a first-line antimalarial regimen in Senegal since 2006. Intermittent preventive treatments with antimalarial drugs based on sulphadoxine-pyrimethamine are also given to children or pregnant women once per month during the transmission season. Since 2006, there have been very few reports on the susceptibility of Plasmodium falciparum to antimalarial drugs. To estimate the prevalence of resistance to several anti-malarial drugs since the introduction of the widespread use of ACT, the presence of molecular markers associated with resistance to chloroquine and sulphadoxine-pyrimethamine was assessed in local isolates at the military hospital of Dakar. METHODS: The prevalence of genetic polymorphisms in genes associated with anti-malarial drug resistance, i.e., Pfcrt, Pfdhfr, Pfdhps and Pfmdr1, and the copy number of Pfmdr1 were evaluated for a panel of 174 isolates collected from patients recruited at the military hospital of Dakar from 14 October 2009 to 19 January 2010. RESULTS: The Pfcrt 76 T mutation was identified in 37.2% of the samples. The Pfmdr1 86Y and 184 F mutations were found in 16.6% and 67.6% of the tested samples, respectively. Twenty-eight of the 29 isolates with the 86Y mutation were also mutated at codon 184. Only one isolate (0.6%) had two copies of Pfmdr1. The Pfdhfr 108 N/T, 51I and 59R mutations were identified in 82.4%, 83.5% and 74.1% of the samples, respectively. The double mutant (108 N and 51I) was detected in 83.5% of the isolates, and the triple mutant (108 N, 51I and 59R) was detected in 75.3%. The Pfdhps 437 G, 436 F/A and 613 S mutations were found in 40.2%, 35.1% and 1.8% of the samples, respectively. There was no double mutant (437 G and 540E) or no quintuple mutant (Pfdhfr 108 N, 51I and 59R and Pfdhps 437 G and 540E). The prevalence of the quadruple mutant (Pfdhfr 108 N, 51I and 59R and Pfdhps 437 G) was 36.5%. CONCLUSIONS: Since 2004, the prevalence of chloroquine resistance had decreased. The prevalence of isolates with high-level pyrimethamine resistance is 83.5%. The prevalence of isolates resistant to sulphadoxine is 40.2%. However, no quintuple mutant (Pfdhfr 108 N, 51I and 59R and Pfdhps 437 G and 540E), which is associated with a high level of sulphadoxine-pyrimethamine resistance, has been identified to date. The resistance to amodiaquine remains moderate.     

Recombination hotspots and population structure in Plasmodium falciparum

Understanding the influences of population structure, selection, and recombination on polymorphism and linkage disequilibrium (LD) is integral to mapping genes contributing to drug resistance or virulence in Plasmodium falciparum. The parasite's short generation time, coupled with a high cross-over rate, can cause rapid LD break-down. However, observations of low genetic variation have led to suggestions of effective clonality: selfing, population admixture, and selection may preserve LD in populations. Indeed, extensive LD surrounding drug-resistant genes has been observed, indicating that recombination and selection play important roles in shaping recent parasite genome evolution. These studies, however, provide only limited information about haplotype variation at local scales. Here we describe the first (to our knowledge) chromosome-wide SNP haplotype and population recombination maps for a global collection of malaria parasites, including the 3D7 isolate, whose genome has been sequenced previously. The parasites are clustered according to continental origin, but alternative groupings were obtained using SNPs at 37 putative transporter genes that are potentially under selection. Geographic isolation and highly variable multiple infection rates are the major factors affecting haplotype structure. Variation in effective recombination rates is high, both among populations and along the chromosome, with recombination hotspots conserved among populations at chromosome ends. This study supports the feasibility of genome-wide association studies in some parasite populations.     

Genetic determination and linkage mapping of Plasmodium falciparum malaria related traits in Senegal

Plasmodium falciparum malaria episodes may vary considerably in their severity and clinical manifestations. There is good evidence that host genetic factors contribute to this variability. To date, most genetic studies aiming at the identification of these genes have used a case/control study design for severe malaria, exploring specific candidate genes. Here, we performed a family-based genetic study of falciparum malaria related phenotypes in two independent longitudinal survey cohorts, as a first step towards the identification of genes and mechanisms involved in the outcome of infection. We studied two Senegalese villages, Dielmo and Ndiop that differ in ethnicity, malaria transmission and endemicity. We performed genome-scan linkage analysis of several malaria-related phenotypes both during clinical attacks and asymptomatic infection. We show evidence for a strong genetic contribution to both the number of clinical falciparum malaria attacks and the asymptomatic parasite density. The asymptomatic parasite density showed linkage to chromosome 5q31 (LOD = 2.26, empirical p = 0.0014, Dielmo), confirming previous findings in other studies. Suggestive linkage values were also obtained at three additional chromosome regions: the number of clinical malaria attacks on chromosome 5p15 (LOD = 2.57, empirical p = 0.001, Dielmo) and 13q13 (LOD = 2.37, empirical p = 0.0014 Dielmo), and the maximum parasite density during asymptomatic infection on chromosome 12q21 (LOD = 3.1, empirical p<10(-4), Ndiop). While regions of linkage show little overlap with genes known to be involved in severe malaria, the four regions appear to overlap with regions linked to asthma or atopy related traits, suggesting that common immune related pathways may be involved.     

The paradoxical population genetics of Plasmodium falciparum

Among the leading causes of death in African children is cerebral malaria caused by the parasitic protozoan Plasmodium falciparum. Endemic forms of this disease are thought to have originated in central Africa 5000-10000 years ago, coincident with the innovation of slash-and-burn agriculture and the diversification of the Anopheles gambiae complex of mosquito vectors. Population genetic studies of P. falciparum have yielded conflicting results. Some evidence suggests that today's population includes multiple ancient lineages pre-dating human speciation. Other evidence suggests that today's population derives from only one, or a small number, of these ancient lineages. Resolution of this issue is important for the evaluation of the long-term efficacy of drug and immunological control strategies.     

Identification of the asymptomatic Plasmodium falciparum and Plasmodium vivax gametocyte reservoir under different transmission intensities

BackgroundUnderstanding epidemiological variables affecting gametocyte carriage and density is essential to design interventions that most effectively reduce malaria human-to-mosquito transmission.Methodology/Principal findingsPlasmodium falciparum and P. vivax parasites and gametocytes were quantified by qPCR and RT-qPCR assays using the same methodologies in 5 cross-sectional surveys involving 16,493 individuals in Brazil, Thailand, Papua New Guinea, and Solomon Islands. The proportion of infections with detectable gametocytes per survey ranged from 44–94% for P. falciparum and from 23–72% for P. vivax. Blood-stage parasite density was the most important predictor of the probability to detect gametocytes. In moderate transmission settings (prevalence by qPCR>5%), parasite density decreased with age and the majority of gametocyte carriers were children. In low transmission settings (prevalence<5%), >65% of gametocyte carriers were adults. Per survey, 37–100% of all individuals positive for gametocytes by RT-qPCR were positive by light microscopy for asexual stages or gametocytes (overall: P. falciparum 178/348, P. vivax 235/398).Conclusions/SignificanceInterventions to reduce human-to-mosquito malaria transmission in moderate-high endemicity settings will have the greatest impact when children are targeted. In contrast, all age groups need to be included in control activities in low endemicity settings to achieve elimination. Detection of infections by light microscopy is a valuable tool to identify asymptomatic blood stage infections that likely contribute most to ongoing transmission at the time of sampling.     

Distribution of IgG subclass antibodies specific for Plasmodium falciparum glutamate-rich-protein molecule in sickle cell trait children with asymptomatic infections

Polymorphism in the beta-globin gene (hemoglobin S) has been associated with protection against severe forms of malaria. In a cross-sectional study, 180 young Gabonese children with and without sickle cell trait and harboring asymptomatic Plasmodium falciparum infections, were assessed for the responses to recombinant protein containing the conserved region of glutamate-rich protein (GLURP). We reported increased age-dependence of antibody prevalence and levels of total IgG (p<0.0001), IgG1 (p=0.009), and IgG3 (p<0.03) antibodies to GLURP with a cut-off at 5 years of age. Whatever the hemoglobin type, cytophilic antibodies (IgG1 and IgG3) were prevalent, but GLURP-specific IgG4 antibodies were detected at significantly (p<0.05) lower levels in HbAS children. We showed that the distribution of non-cytophilic IgG antibodies differs according to the hemoglobin type and to the malaria antigens tested. This may have possible implication for the clearance of malaria parasites and for protection against severe malaria.     

Impact of red blood cell polymorphisms on the antibody response to Plasmodium falciparum in Senegal

The evidence of protection afforded by red blood cell polymorphisms against either clinical malaria or Plasmodium falciparum blood levels varies with the study site and the type of malaria transmission. Nevertheless, no clear implication of an antibody-related effect has yet been established in the protection related to red blood cell polymorphisms. We performed a prospective study, where plasma IgG and IgG subclasses directed to recombinant proteins from the merozoite surface protein 2 (MSP2/3D7 and MSP2/FC27) and the ring-infected erythrocyte surface antigen (RESA) were determined in a cohort of 413 Senegalese children before the annual malaria transmission season. The antibody response was dependent on age, and to a lesser extent, on the village of residence. IgG3 responders to all proteins, IgG responders to RESA and MSP2/3D7, as well as IgG2 to RESA and IgG1 responders to MSP2/3D7, presented enhanced mean values of parasite density, as evaluated during an 18-month follow-up. The levels of IgG and IgG3 to MSP2/3D7 were negatively associated with the risk of occurrence of a malaria attack during the following transmission season. Compared to normal children, sickle cell trait carriers presented lower levels of IgG to MSP2/3D7. Similarly, G6PD A- girls had lower levels of IgG and IgG3 to MSP2/FC27 than did G6PD normal girls. The impact of these particular genetic polymorphisms on the modulation of the antibody response is discussed.     

High prevalence of Plasmodium malariae and Plasmodium ovale in co-infections with Plasmodium falciparum in asymptomatic malaria parasite carriers in southwestern Nigeria

Asymptomatic malaria parasite carriers do not seek anti-malarial treatment and may constitute a silent infectious reservoir. In order to assess the level of asymptomatic and symptomatic carriage amongst adolescents in a highly endemic area, and to identify the risk factors associated with such carriage, we conducted a cross-sectional survey of 1032 adolescents (ages 10–19 years) from eight schools located in Ibadan, southwestern Nigeria in 2016. Blood films and blood spot filter paper samples were prepared for microscopy and DNA analysis. The prevalence of asymptomatic malaria was determined using microscopy, rapid diagnostic tests and PCR for 658 randomly selected samples. Of these, we found that 80% of asymptomatic schoolchildren were positive for malaria parasites by PCR, compared with 47% and 9%, determined by rapid diagnostic tests and microscopy, respectively. Malaria parasite species typing was performed using PCR targeting the mitochondrial CoxIII gene, and revealed high rates of carriage of Plasmodium malariae (53%) and Plasmodium ovale (24%). Most asymptomatic infections were co-infections of two or more species (62%), with Plasmodium falciparum + P. malariae the most common (35%), followed by P. falciparum + P. malariae + P. ovale (21%) and P. falciparum + P. ovale (6%). Single infections of P. falciparum, P. malariae and P. ovale accounted for 24%, 10% and 4% of all asymptomatic infections, respectively. To compare the species composition of asymptomatic and symptomatic infections, further sample collection was carried out in 2017 at one of the previously sampled schools, and at a nearby hospital. Whilst the species composition of the asymptomatic infections was similar to that observed in 2016, the symptomatic infections were markedly different, with single infections of P. falciparum observed in 91% of patients, P. falciparum + P. malariae in 5% and P. falciparum + P. ovale in 4%.     

Longitudinal study of Plasmodium falciparum and Plasmodium vivax in a Karen population in Thailand

Background

Pregnancy malaria is caused by Plasmodium falciparum -infected erythrocytes binding the placental receptor chondroitin sulfate A (CSA). This results in accumulation of parasites in the placenta with severe clinical consequences for the mother and her unborn child. Women become resistant to placental malaria as antibodies are acquired which specifically target the surface of infected erythrocytes binding in the placenta. VAR2CSA is most likely the parasite-encoded protein which mediates binding to the placental receptor CSA. Several domains have been shown to bind CSA in vitro; and it is apparent that a VAR2CSA-based vaccine cannot accommodate all the CSA binding domains and serovariants. It is thus of high priority to define minimal ligand binding regions throughout the VAR2CSA molecule.

Methods

To define minimal CSA-binding regions/peptides of VAR2CSA, a phage display library based on the entire var2csa coding region was constructed. This library was screened on immobilized CSA and cells expressing CSA resulting in a limited number of CSA-binding phages. Antibodies against these peptides were affinity purified and tested for reactivity against CSA-binding infected erythrocytes.

Results

The most frequently identified phages expressed peptides residing in the parts of VAR2CSA previously defined as CSA binding. In addition, most of the binding regions mapped to surface-exposed parts of VAR2CSA. The binding of a DBL2X peptide to CSA was confirmed with a synthetic peptide. Antibodies against a CSA-binding DBL2X peptide reacted with the surface of infected erythrocytes indicating that this epitope is accessible for antibodies on native VAR2CSA on infected erythrocytes.

Conclusion

Short continuous regions of VAR2CSA with affinity for multiple types of CSA were defined. A number of these regions localize to CSA-binding domains and to surface-exposed regions within these domains and a synthetic peptide corresponding to a peptide sequence in DBL2 was shown to bind to CSA and not to CSC. It is likely that some of these epitopes are involved in native parasite CSA adhesion. However, antibodies directed against single epitopes did not inhibit parasite adhesion. This study supports phage display as a technique to identify CSA-binding regions of large proteins such as VAR2CSA.