Understanding the significance of oxygen tension on the biology of Plasmodium falciparum blood stages: From the human body to the laboratory.

Plasmodium falciparum undergoes sequestration within deep tissues of the human body, spanning multiple organ systems with differing oxygen (O2) concentrations. The parasite is exposed to an even greater range of O2 concentrations as it transitions from the human to the mosquito host, suggesting a high level of plasticity as it navigates these different environments. In this review, we explore factors that may contribute to the parasite's response to different environmental O2 concentrations, recognizing that there are likely multiple pieces to this puzzle. We first review O2-sensing mechanisms, which exist in other apicomplexans such as Toxoplasma gondii and consider whether similar systems could exist in Plasmodium. Next, we review morphological and functional changes in P. falciparum's mitochondrion during the asexual-to-sexual stage transition and discuss how these changes overlap with the parasite's access to O2. We then delve into reactive oxygen species (ROS) as ROS production is influenced by O2 availability and oxidative stress impacts Plasmodium intraerythrocytic development. Lastly, given that the primary role of the red blood cell (RBC) is to deliver O2 throughout the body, we discuss how changes in the oxygenation status of hemoglobin, the RBC's O2-carrying protein and key nutrient for Plasmodium, could also potentially impact the parasite's growth during intraerythrocytic development. This review also highlights studies that have investigated P. falciparum biology under varying O2 concentrations and covers technical aspects related to P. falciparum cultivation in the lab, focusing on sources of technical variation that could alter the amount of dissolved O2 encountered by cells during in vitro experiments. Lastly, we discuss how culture systems can better replicate in vivo heterogeneity with respect to O2 gradients, propose ideas for further research in this area, and consider translational implications related to O2 and malaria.

Two mosquito salivary antigens demonstrate promise as biomarkers of recent exposure to P. falciparum infected mosquito bites.

BACKGROUND: Measuring malaria transmission intensity using the traditional entomological inoculation rate is difficult. Antibody responses to mosquito salivary proteins like SG6 have been used as biomarkers of exposure to Anopheles mosquito bites. Here, we investigate four mosquito salivary proteins as potential biomarkers of human exposure to mosquitoes infected with P. falciparum: mosGILT, SAMSP1, AgSAP, and AgTRIO. METHODS: We tested population-level human immune responses in longitudinal and cross-sectional plasma from individuals with known P. falciparum infection from low and moderate transmission areas in Senegal using a multiplexed magnetic bead-based assay. RESULTS: AgSAP and AgTRIO were the best indicators of recent exposure to infected mosquitoes. Antibody responses to AgSAP, in a moderate endemic area, and to AgTRIO in both low and moderate endemic areas, were significantly higher than responses in a healthy non-endemic control cohort (p-values = 0.0245, 0.0064, and <0.0001 respectively). No antibody responses significantly differed between the low and moderate transmission area, or between equivalent groups during and outside the malaria transmission seasons. For AgSAP and AgTRIO, reactivity peaked 2-4 weeks after clinical P. falciparum infection and declined 3 months after infection. DISCUSSION: Reactivity to AgSAP and AgTRIO peaked after infection, with no differences between transmission seasons within region or between low and moderate transmission regions. This suggests that reactivity reflects exposure to infectious mosquitoes or recent bites rather than general mosquito exposure. Kinetics suggest reactivity is relatively short-lived. AgSAP and AgTRIO are promising candidates to incorporate into multiplexed assays for serosurveillance of population-level changes in P. falciparum-infected mosquito exposure.

Two decades of molecular surveillance in Senegal reveal rapid changes in known drug resistance mutations over time.

BACKGROUND: Drug resistance in Plasmodium falciparum is a major threat to malaria control efforts. Pathogen genomic surveillance could be invaluable for monitoring current and emerging parasite drug resistance. METHODS: Data from two decades (2000-2020) of continuous molecular surveillance of P. falciparum parasites from Senegal were retrospectively examined to assess historical changes in malaria drug resistance mutations. Several known drug resistance markers and their surrounding haplotypes were profiled using a combination of single nucleotide polymorphism (SNP) molecular surveillance and whole genome sequence based population genomics. RESULTS: This dataset was used to track temporal changes in drug resistance markers whose timing correspond to historically significant events such as the withdrawal of chloroquine (CQ) and the introduction of sulfadoxine-pyrimethamine (SP) in 2003. Changes in the mutation frequency at Pfcrt K76T and Pfdhps A437G coinciding with the 2014 introduction of seasonal malaria chemoprevention (SMC) in Senegal were observed. In 2014, the frequency of Pfcrt K76T increased while the frequency of Pfdhps A437G declined. Haplotype-based analyses of Pfcrt K76T showed that this rapid increase was due to a recent selective sweep that started after 2014. DISCUSSION (CONCLUSION): The rapid increase in Pfcrt K76T is troubling and could be a sign of emerging amodiaquine (AQ) resistance in Senegal. Emerging AQ resistance may threaten the future clinical efficacy of artesunate-amodiaquine (ASAQ) and AQ-dependent SMC chemoprevention. These results highlight the potential of molecular surveillance for detecting rapid changes in parasite populations and stress the need to monitor the effectiveness of AQ as a partner drug for artemisinin-based combination therapy (ACT) and for chemoprevention.

Mapping partner drug resistance to guide antimalarial combination therapy policies in sub-Saharan Africa.

Resistance to artemisinin-based combination therapies (ACTs) threatens the global control of Plasmodium falciparum malaria. ACTs combine artemisinin-derived compounds with partner drugs to enable multiple mechanisms of clearance. Although ACTs remain widely effective in sub-Saharan Africa, long-standing circulation of parasite alleles associated with reduced partner drug susceptibility may contribute to the development of clinical resistance. We fitted a hierarchical Bayesian spatial model to data from over 500 molecular surveys to predict the prevalence and frequency of four key markers in transporter genes (pfcrt 76T and pfmdr1 86Y, 184F, and 1246Y) in first-level administrative divisions in sub-Saharan Africa from the uptake of ACTs (2004 to 2009) to their widespread usage (2010 to 2018). Our models estimated that the pfcrt 76T mutation decreased in prevalence in 90% of regions; the pfmdr1 N86 and D1246 wild-type genotypes increased in prevalence in 96% and 82% of regions, respectively; and there was no significant directional selection at the pfmdr1 Y184F locus. Rainfall seasonality was the strongest predictor of the prevalence of wild-type genotypes, with other covariates, including first-line drug policy and transmission intensity more weakly associated. We lastly identified regions of high priority for enhanced surveillance that could signify decreased susceptibility to the local first-line ACT. Our results can be used to infer the degree of molecular resistance and magnitude of wild-type reversion in regions without survey data to inform therapeutic policy decisions.

Accounting for red blood cell accessibility reveals distinct invasion strategies in Plasmodium falciparum strains.

The growth of the malaria parasite Plasmodium falciparum in human blood causes all the symptoms of malaria. To proliferate, non-motile parasites must have access to susceptible red blood cells, which they invade using pairs of parasite ligands and host receptors that define invasion pathways. Parasites can switch invasion pathways, and while this flexibility is thought to facilitate immune evasion, it may also reflect the heterogeneity of red blood cell surfaces within and between hosts. Host genetic background affects red blood cell structure, for example, and red blood cells also undergo dramatic changes in morphology and receptor density as they age. The in vivo consequences of both the accessibility of susceptible cells, and their heterogeneous susceptibility, remain unclear. Here, we measured invasion of laboratory strains of P. falciparum relying on distinct invasion pathways into red blood cells of different ages. We estimated invasion efficiency while accounting for red blood cell accessibility to parasites. This approach revealed different tradeoffs made by parasite strains between the fraction of cells they can invade and their invasion rate into them, and we distinguish "specialist" strains from "generalist" strains in this context. We developed a mathematical model to show that generalist strains would lead to higher peak parasitemias in vivo compared to specialist strains with similar overall proliferation rates. Thus, the ecology of red blood cells may play a key role in determining the rate of P. falciparum parasite proliferation and malaria virulence.

Genetic diversity of Plasmodium falciparum in Grande Comore Island.

BACKGROUND: Despite several control interventions resulting in a considerable decrease in malaria prevalence in the Union of the Comoros, the disease remains a public health problem with high transmission in Grande Comore compared to neighbouring islands. In this country, only a few studies investigating the genetic diversity of Plasmodium falciparum have been performed so far. For this reason, this study aims to examine the genetic diversity of P. falciparum by studying samples collected in Grande Comore in 2012 and 2013, using merozoite surface protein 1 (msp1), merozoite surface protein 2 (msp2) and single nucleotide polymorphism (SNP) genetic markers. METHODS: A total of 162 positive rapid diagnostic test (RDT) samples from Grande Comore were used to extract parasite DNA. Allelic families K1, Mad20 and RO33 of the msp1 gene as well as allelic families IC3D7 and FC37 of the msp2 gene were determined by using nested PCR. Additionally, 50 out of 151 samples were genotyped to study 24 SNPs by using high resolution melting (HRM). RESULTS: Two allelic families were predominant, the K1 family of msp1 gene (55%) and the FC27 family of msp2 gene (47.4%). Among 50 samples genotyped for 24 SNPs, 42 (84%) yielded interpretable results. Out of these isolates, 36 (85%) were genetically unique and 6 (15%) grouped into two clusters. The genetic diversity of P. falciparum calculated from msp1 and msp2 genes and SNPs was 0.82 and 0.61, respectively. CONCLUSION: In summary, a large genetic diversity of P. falciparum was observed in Grande Comore. This may favour persistence of malaria and might be one of the reasons for the high malaria transmission compared to neighbouring islands. Further surveillance of P. falciparum isolates, mainly through environmental management and vector control, is warranted until complete elimination is attained.

Molecular epidemiology of Plasmodium falciparum by multiplexed amplicon deep sequencing in Senegal.

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.

Genetic evidence for imported malaria and local transmission in Richard Toll, Senegal.

BACKGROUND: Malaria elimination efforts can be undermined by imported malaria infections. Imported infections are classified based on travel history. METHODS: A genetic strategy was applied to better understand the contribution of imported infections and to test for local transmission in the very low prevalence region of Richard Toll, Senegal. RESULTS: Genetic relatedness analysis, based upon molecular barcode genotyping data derived from diagnostic material, provided evidence for both imported infections and ongoing local transmission in Richard Toll. Evidence for imported malaria included finding that a large proportion of Richard Toll parasites were genetically related to parasites from Thiès, Senegal, a region of moderate transmission with extensive available genotyping data. Evidence for ongoing local transmission included finding parasites of identical genotype that persisted across multiple transmission seasons as well as enrichment of highly related infections within the households of non-travellers compared to travellers. CONCLUSIONS: These data indicate that, while a large number of infections may have been imported, there remains ongoing local malaria transmission in Richard Toll. These proof-of-concept findings underscore the value of genetic data to identify parasite relatedness and patterns of transmission to inform optimal intervention selection and placement.

De Novo Mutations Resolve Disease Transmission Pathways in Clonal Malaria.

Detecting de novo mutations in viral and bacterial pathogens enables researchers to reconstruct detailed networks of disease transmission and is a key technique in genomic epidemiology. However, these techniques have not yet been applied to the malaria parasite, Plasmodium falciparum, in which a larger genome, slower generation times, and a complex life cycle make them difficult to implement. Here, we demonstrate the viability of de novo mutation studies in P. falciparum for the first time. Using a combination of sequencing, library preparation, and genotyping methods that have been optimized for accuracy in low-complexity genomic regions, we have detected de novo mutations that distinguish nominally identical parasites from clonal lineages. Despite its slower evolutionary rate compared with bacterial or viral species, de novo mutation can be detected in P. falciparum across timescales of just 1-2 years and evolutionary rates in low-complexity regions of the genome can be up to twice that detected in the rest of the genome. The increased mutation rate allows the identification of separate clade expansions that cannot be found using previous genomic epidemiology approaches and could be a crucial tool for mapping residual transmission patterns in disease elimination campaigns and reintroduction scenarios.

Temporal changes in Plasmodium falciparum reticulocyte binding protein homolog 2b (PfRh2b) in Senegal and The Gambia.

BACKGROUND: The Plasmodium falciparum reticulocyte binding protein homolog 2b (PfRh2b) is an important P. falciparum merozoite ligand that mediates invasion of erythrocytes by interacting with a chymotrypsin-sensitive "receptor Z". A large deletion polymorphism is found in the c-terminal ectodomain of this protein in many countries around the world, resulting in a truncated, but expressed protein. The varying frequencies by region suggest that there could be region specific immune selection at this locus. Therefore, this study was designed to determine temporal changes in the PfRh2b deletion polymorphism in infected individuals from Thiès (Senegal) and Western Gambia (The Gambia). It was also sought to determine the selective pressures acting at this locus and whether prevalence of the deletion in isolates genotyped by a 24-SNP molecular barcode is linked to background genotype or whether there might be independent selection acting at this locus. METHODS: Infected blood samples were sourced from archives of previous studies conducted between 2007 and 2013 at SLAP clinic in Thiès and from 1984 to 2013 in Western Gambia by MRC Unit at LSHTM, The Gambia. A total of 1380 samples were screened for the dimorphic alleles of the PfRh2b using semi-nested Polymerase Chain Reaction PCR. Samples from Thiès were previously barcoded. RESULTS: In Thiès, a consistent trend of decreasing prevalence of the PfRh2b deletion over time was observed: from 66.54% in 2007 and to 38.1% in 2013. In contrast, in Western Gambia, the frequency of the deletion fluctuated over time; it increased between 1984 and 2005 from (58.04%) to (69.33%) and decreased to 47.47% in 2007. Between 2007 and 2012, the prevalence of this deletion increased significantly from 47.47 to 83.02% and finally declined significantly to 57.94% in 2013. Association between the presence of this deletion and age was found in Thiès, however, not in Western Gambia. For the majority of isolates, the PfRh2b alleles could be tracked with specific 24-SNP barcoded genotype, indicating a lack of independent selection at this locus. CONCLUSION: PfRh2b deletion was found in the two countries with varying prevalence during the study period. However, these temporal and spatial variations could be an obstacle to the implementation of this protein as a potential vaccine candidate.

Dramatic Changes in Malaria Population Genetic Complexity in Dielmo and Ndiop, Senegal, Revealed Using Genomic Surveillance.

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

Functional Analysis Reveals Geographical Variation in Inhibitory Immune Responses Against a Polymorphic Malaria Antigen.

Background: Plasmodium falciparum reticulocyte-binding protein homologue 2b (PfRh2b) is an invasion ligand that is a potential blood-stage vaccine candidate antigen; however, a naturally occurring deletion within an immunogenic domain is present at high frequencies in Africa and has been associated with alternative invasion pathway usage. Standardized tools that provide antigenic specificity in in vitro assays are needed to functionally assess the neutralizing potential of humoral responses against malaria vaccine candidate antigens. Methods: Transgenic parasite lines were generated to express the PfRh2b deletion. Total immunoglobulin G (IgG) from individuals residing in malaria-endemic regions in Tanzania, Senegal, and Mali were used in growth inhibition assays with transgenic parasite lines. Results: While the PfRh2b deletion transgenic line showed no change in invasion pathway utilization compared to the wild-type in the absence of specific antibodies, it outgrew wild-type controls in competitive growth experiments. Inhibition differences with total IgG were observed in the different endemic sites, ranging from allele-specific inhibition to allele-independent inhibitory immune responses. Conclusions: The PfRh2b deletion may allow the parasite to escape neutralizing antibody responses in some regions. This difference in geographical inhibition was revealed using transgenic methodologies, which provide valuable tools for functionally assessing neutralizing antibodies against vaccine-candidate antigens in regions with varying malaria endemicity.

Continued Transmission of Zika Virus in Humans in West Africa, 1992-2016.

First identified in 1947 in Uganda, Zika virus (ZIKV) has remained largely unstudied until the recent outbreak in Latin America. This study aimed to measure the prevalence of ZIKV in febrile patients in Senegal and Nigeria in samples collected from 1992 to 2016. The seroprevalence of ZIKV was 6.2% based on ZIKV immunoglobulin M and negative for dengue reactivity. ZIKV envelope was amplified from 4 samples. Phylogenetic analysis showed that the ZIKVs belonged to the African lineage, grouping with either the Nigerian or MR766 sublineages. This study provides evidence that ZIKV has been silently circulating in West Africa for 2 decades.

Genetic Evidence for Erythrocyte Receptor Glycophorin B Expression Levels Defining a Dominant Plasmodium falciparum Invasion Pathway into Human Erythrocytes.

Plasmodium falciparum, the parasite that causes the deadliest form of malaria, has evolved multiple proteins known as invasion ligands that bind to specific erythrocyte receptors to facilitate invasion of human erythrocytes. The EBA-175/glycophorin A (GPA) and Rh5/basigin ligand-receptor interactions, referred to as invasion pathways, have been the subject of intense study. In this study, we focused on the less-characterized sialic acid-containing receptors glycophorin B (GPB) and glycophorin C (GPC). Through bioinformatic analysis, we identified extensive variation in glycophorin B (GYPB) transcript levels in individuals from Benin, suggesting selection from malaria pressure. To elucidate the importance of the GPB and GPC receptors relative to the well-described EBA-175/GPA invasion pathway, we used an ex vivo erythrocyte culture system to decrease expression of GPA, GPB, or GPC via lentiviral short hairpin RNA transduction of erythroid progenitor cells, with global surface proteomic profiling. We assessed the efficiency of parasite invasion into knockdown cells using a panel of wild-type P. falciparum laboratory strains and invasion ligand knockout lines, as well as P. falciparum Senegalese clinical isolates and a short-term-culture-adapted strain. For this, we optimized an invasion assay suitable for use with small numbers of erythrocytes. We found that all laboratory strains and the majority of field strains tested were dependent on GPB expression level for invasion. The collective data suggest that the GPA and GPB receptors are of greater importance than the GPC receptor, supporting a hierarchy of erythrocyte receptor usage in P. falciparum.

Factors influencing the use of malaria prevention strategies by women in Senegal: a cross-sectional study.

BACKGROUND: The World Health Organization (WHO) recommends the use of insecticide-treated nets (ITNs) and intermittent preventive treatment in pregnancy (IPTp) as a cost-effective intervention for the prevention of malaria during pregnancy in endemic areas. This study was conducted to investigate: (1) the extent of use of both IPTp and ITNs, and (2) conduct multinomial regression to identify factors affecting the optimal usage of IPTp and ITNs among women with a recent pregnancy in Senegal. METHODS: Data was drawn from the 2013-2014 Demographic and Health Survey. A total of 4616 women aged 15-49 years old, who had a recent pregnancy were analyzed. Multinomial logistic regression model was used to assess factors associated with optimal uptake of malaria preventive strategies (both IPTp and ITN use). RESULTS: Amongst women who had a recent pregnancy, less than half of them used ITNs (46.84%) however, 80.35% reported taking IPTp during their last pregnancy. Overall, 37.51% reported using the optimal malaria preventive strategies. Women aged 35-49 years and living in the richer or middle wealth quintile were more likely to use optimal prevention methods. Pregnant women living in Diourbel, Saint-Louis, Thies, Louga, Fatick and Matam were more likely to use both IPTp-SP and ITNs compared to those living in Dakar. Additionally, women who initiated antenatal care in at least at 6 weeks of pregnancy or who attended four antenatal visits or more were more likely to use optimal malaria preventive methods during pregnancy. CONCLUSIONS: This study has shown important factors that influence the uptake of malaria prevention methods during pregnancy in Senegal. These findings highlight the need for targeted preventive strategies when designing and implementing policies aimed at improving the uptake of these measures during pregnancy in Senegal.

Ex vivo susceptibility and genotyping of Plasmodium falciparum isolates from Pikine, Senegal.

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.

High resolution melting: a useful field-deployable method to measure dhfr and dhps drug resistance in both highly and lowly endemic Plasmodium populations.

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.

Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum.

Erythrocyte invasion by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion requires a series of extracellular recognition events between erythrocyte receptors and ligands on the merozoite, the invasive form of the parasite. None of the few known receptor-ligand interactions involved are required in all parasite strains, indicating that the parasite is able to access multiple redundant invasion pathways. Here, we show that we have identified a receptor-ligand pair that is essential for erythrocyte invasion in all tested P. falciparum strains. By systematically screening a library of erythrocyte proteins, we have found that the Ok blood group antigen, basigin, is a receptor for PfRh5, a parasite ligand that is essential for blood stage growth. Erythrocyte invasion was potently inhibited by soluble basigin or by basigin knockdown, and invasion could be completely blocked using low concentrations of anti-basigin antibodies; importantly, these effects were observed across all laboratory-adapted and field strains tested. Furthermore, Ok(a-) erythrocytes, which express a basigin variant that has a weaker binding affinity for PfRh5, had reduced invasion efficiencies. Our discovery of a cross-strain dependency on a single extracellular receptor-ligand pair for erythrocyte invasion by P. falciparum provides a focus for new anti-malarial therapies.

The Clinically Tested Gardos Channel Inhibitor Senicapoc Exhibits Antimalarial Activity.

Senicapoc, a Gardos channel inhibitor, prevented erythrocyte dehydration in clinical trials of patients with sickle cell disease. We tested the hypothesis that senicapoc-induced blockade of the Gardos channel inhibits Plasmodium growth. Senicapoc inhibited in vitro growth of human and primate plasmodia during the clinical blood stage. Senicapoc treatment suppressed P. yoelii parasitemia in vivo in C57BL/6 mice. The reassuring safety and biochemical profile of senicapoc encourage its use in antimalarial development.

Selection of N86F184D1246 haplotype of Pfmrd1 gene by artemether-lumefantrine drug pressure on Plasmodium falciparum populations in Senegal.

BACKGROUND: The use of artemisinin as a monotherapy resulted in the emergence of artemisinin resistance in 2005 in Southeast Asia. Monitoring of artemisinin combination therapy (ACT) is critical in order to detect and prevent the spread of resistance in endemic areas. Ex vivo studies and genotyping of molecular markers of resistance can be used as part of this routine monitoring strategy. One gene that has been associated in some ACT partner drug resistance is the Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene. The purpose of this study was to assess the drug susceptibility of P. falciparum populations from Thiès, Senegal by ex vivo assay and typing molecular markers of resistance to drug components of ACT currently used for treatment. METHODS: The ex vivo susceptibility of 170 P. falciparum isolates to chloroquine, amodiaquine, lumefantrine, artesunate, and artemether was determined using the DAPI ex vivo assay. The high resolution melting technique was used to genotype the pfmdr1 gene at codons 86, 184 and 1246. RESULTS: A significant decrease in IC50 values was observed between 2012 and 2013: from 13.84 to 6.484 for amodiaquine, 173.4 to 113.2 for lumefantrine, and 39.72 to 18.29 for chloroquine, respectively. Increase of the wild haplotype NYD and the decrease of the mutant haplotype NFD (79 and 62.26 %) was also observed. A correlation was observed between the wild type allele Y184 in pfmdr1 and higher IC50 for all drugs, except amodiaquine. CONCLUSION: This study has shown an increase in sensitivity over the span of two transmission seasons, marked by an increase in the WT alleles at pfmdr1. Continuous the monitoring of the ACT used for treatment of uncomplicated malaria will be helpful.