The Prevalence of Human Plasmodium Species during Peak Transmission Seasons from 2016 to 2021 in the Rural Commune of Ntjiba, Mali.

Up-to-date knowledge of key epidemiological aspects of each Plasmodium species is necessary for making informed decisions on targeted interventions and control strategies to eliminate each of them. This study aims to describe the epidemiology of plasmodial species in Mali, where malaria is hyperendemic and seasonal. Data reports collected during high-transmission season over six consecutive years were analyzed to summarize malaria epidemiology. Malaria species and density were from blood smear microscopy. Data from 6870 symptomatic and 1740 asymptomatic participants were analyzed. The median age of participants was 12 years, and the sex ratio (male/female) was 0.81. Malaria prevalence from all Plasmodium species was 65.20% (95% CI: 60.10-69.89%) and 22.41% (CI: 16.60-28.79%) for passive and active screening, respectively. P. falciparum was the most prevalent species encountered in active and passive screening (59.33%, 19.31%). This prevalence was followed by P. malariae (1.50%, 1.15%) and P. ovale (0.32%, 0.06%). Regarding frequency, P. falciparum was more frequent in symptomatic individuals (96.77% vs. 93.24%, p = 0.014). In contrast, P. malariae was more frequent in asymptomatic individuals (5.64% vs. 2.45%, p < 0.001). P. ovale remained the least frequent species (less than 1%), and no P. vivax was detected. The most frequent coinfections were P. falciparum and P. malariae (0.56%). Children aged 5-9 presented the highest frequency of P. falciparum infections (41.91%). Non-falciparum species were primarily detected in adolescents (10-14 years) with frequencies above 50%. Only P. falciparum infections had parasitemias greater than 100,000 parasites per µL of blood. P. falciparum gametocytes were found with variable prevalence across age groups. Our data highlight that P. falciparum represented the first burden, but other non-falciparum species were also important. Increasing attention to P. malariae and P. ovale is essential if malaria elimination is to be achieved.

Plasmodium falciparum Development from Gametocyte to Oocyst: Insight from Functional Studies.

Malaria elimination may never succeed without the implementation of transmission-blocking strategies. The transmission of Plasmodium spp. parasites from the human host to the mosquito vector depends on circulating gametocytes in the peripheral blood of the vertebrate host. Once ingested by the mosquito during blood meals, these sexual forms undergo a series of radical morphological and metabolic changes to survive and progress from the gut to the salivary glands, where they will be waiting to be injected into the vertebrate host. The design of effective transmission-blocking strategies requires a thorough understanding of all the mechanisms that drive the development of gametocytes, gametes, sexual reproduction, and subsequent differentiation within the mosquito. The drastic changes in Plasmodium falciparum shape and function throughout its life cycle rely on the tight regulation of stage-specific gene expression. This review outlines the mechanisms involved in Plasmodium falciparum sexual stage development in both the human and mosquito vector, and zygote to oocyst differentiation. Functional studies unravel mechanisms employed by P. falciparum to orchestrate the expression of stage-specific functional products required to succeed in its complex life cycle, thus providing us with potential targets for developing new therapeutics. These mechanisms are based on studies conducted with various Plasmodium species, including predominantly P. falciparum and the rodent malaria parasites P. berghei. However, the great potential of epigenetics, genomics, transcriptomics, proteomics, and functional genetic studies to improve the understanding of malaria as a disease remains partly untapped because of limitations in studies using human malaria parasites and field isolates.

IFNγ, TNFα polymorphisms and IFNγ serum levels are associated with the clearance of drug-resistant P. falciparum in Malian children.

Host immunity has been suggested to clear drug-resistant parasites in malaria-endemic settings. However, the immunogenetic mechanisms involved in parasite clearance are poorly understood. Characterizing the host's immunity and genes involved in controlling the parasitic infection can inform the development of blood-stage malaria vaccines. This study investigates host regulatory cytokines and immunogenomic factors associated with the clearance of Plasmodium falciparum carrying a chloroquine resistance genotype. Biological samples from participants of previous drug efficacy trials conducted in two Malian localities were retrieved. The P. falciparum chloroquine resistance transporter (Pfcrt) gene was genotyped using parasite DNA. Children carrying parasites with the mutant allele (Pfcrt-76T) were classified based on their ability to clear their parasites. The levels of the different cytokines were measured in serum. The polymorphisms of specific human genes involved in malaria susceptibility were genotyped using human DNA. The prevalence of the Pfcrt-76T was significantly higher in Kolle than in Bandiagara (81.6 % vs 38.6 %, p < 10-6). The prevalence of children who cleared their mutant parasites was significantly higher in Bandiagara than in Kolle (82.2 % vs 67.4 %, p < 0.05). The genotyping of host genes revealed that IFN-γ -874 T and TNF-α -308A alleles were positively associated with parasite clearance. Cytokine profiling revealed that IFN-γ level was positively associated with parasite clearance (p = 0.04). This study highlights the role of host's immunity and immunogenetic factors to clear resistant parasites, suggesting further characterization of these polymorphisms may help to develop novel approaches to antiparasitic treatment strategies.

A Novel Ex Vivo Drug Assay for Assessing the Transmission-Blocking Activity of Compounds on Field-Isolated Plasmodium falciparum Gametocytes.

The discovery and development of transmission-blocking therapies challenge malaria elimination and necessitate standard and reproducible bioassays to measure the blocking properties of antimalarial drugs and candidate compounds. Most of the current bioassays evaluating the transmission-blocking activity of compounds rely on laboratory-adapted Plasmodium strains. Transmission-blocking data from clinical gametocyte isolates could help select novel transmission-blocking candidates for further development. Using freshly collected Plasmodium falciparum gametocytes from asymptomatic individuals, we first optimized ex vivo culture conditions to improve gametocyte viability and infectiousness by testing several culture parameters. We next pre-exposed ex vivo field-isolated gametocytes to chloroquine, dihydroartemisinin, primaquine, KDU691, GNF179, and oryzalin for 48 h prior to direct membrane feeding. We measured the activity of the drug on the ability of gametocytes to resume the sexual life cycle in Anopheles after drug exposure. Using 57 blood samples collected from Malian volunteers aged 6 to 15 years, we demonstrate that the infectivity of freshly collected field gametocytes can be preserved and improved ex vivo in a culture medium supplemented with 10% horse serum at 4% hematocrit for 48 h. Moreover, our optimized drug assay displays the weak transmission-blocking activity of chloroquine and dihydroartemisinin, while primaquine and oryzalin exhibited a transmission-blocking activity of ~50% at 1 µM. KDU691 and GNF179 both interrupted Plasmodium transmission at 1 µM and 5 nM, respectively. This new approach, if implemented, has the potential to accelerate the screening of compounds with transmission-blocking activity.

Hands-on training in structural biology, a tool for sustainable development in Africa series 4.

Structural biology is an essential tool for understanding the molecular basis of diseases, which can guide the rational design of new drugs, vaccines, and the optimisation of existing medicines. However, most African countries do not conduct structural biology research due to limited resources, lack of trained persons, and an exodus of skilled scientists. The most urgent requirement is to build on the emerging centres in Africa - some well-established, others growing. This can be achieved through workshops that improve networking, grow skills, and develop mechanisms for access to light source beamlines for defining X-ray structures across the continent. These would encourage the growth of structural biology, which is central to understanding biological functions and developing new antimicrobials and other drugs. In this light, a hands-on training workshop in structural biology series 4 was organised by BioStruct-Africa and the Malaria Research and Training Center (MRTC) in Bamako, Mali, to help bridge this gap. The workshop was hosted by MRTC from the 25th to 28th of April 2022. Through a series of lectures and practicals, the workshop enlightened the participants on how structural biology can be utilised to find solutions to the prevalent diseases in Africa. The short training gave them an overview of target selection, protein production and purification, structural determination techniques, and analysis in combination with high-throughput, structure-guided, fragment-based drug design.

Uptake of Plasmodium falciparum Gametocytes During Mosquito Bloodmeal by Direct and Membrane Feeding.

Plasmodium falciparum remains one of the leading causes of child mortality, and nearly half of the world's population is at risk of contracting malaria. While pathogenesis results from replication of asexual forms in human red blood cells, it is the sexually differentiated forms, gametocytes, which are responsible for the spread of the disease. For transmission to succeed, both mature male and female gametocytes must be taken up by a female Anopheles mosquito during its blood meal for subsequent differentiation into gametes and mating inside the mosquito gut. Observed circulating numbers of gametocytes in the human host are often surprisingly low. A pre-fertilization behavior, such as skin sequestration, has been hypothesized to explain the efficiency of human-to-mosquito transmission but has not been sufficiently tested due to a lack of appropriate tools. In this study, we describe the optimization of a qPCR tool that enables the relative quantification of gametocytes within very small input samples. Such a tool allows for the quantification of gametocytes in different compartments of the host and the vector that could potentially unravel mechanisms that enable highly efficient malaria transmission. We demonstrate the use of our gametocyte quantification method in mosquito blood meals from both direct skin feeding on Plasmodium gametocyte carriers and standard membrane feeding assay. Relative gametocyte abundance was not different between mosquitoes fed through a membrane or directly on the skin suggesting that there is no systematic enrichment of gametocytes picked up in the skin.

Differential infectivity of gametocytes after artemisinin-based combination therapy of uncomplicated falciparum malaria.

BACKGROUND: Most malaria-endemic countries use artemisinin-based combination therapy (ACT) as their first-line treatment. ACTs are known to be highly effective on asexual stages of the malaria parasite. Malaria transmission and the spread of resistant parasites depend on the infectivity of gametocytes. The effect of the current ACT regimens on gametocyte infectivity is unclear. OBJECTIVES: This study aimed to determine the infectivity of gametocytes to Anopheles gambiae following ACT treatment in the field. METHODS: During a randomised controlled trial in Bougoula-Hameau, Mali, conducted from July 2005 to July 2007, volunteers with uncomplicated malaria were randomised to receive artemether-lumefantrine, artesunate-amodiaquine, or artesunate-sulfadoxine/pyrimethamine. Volunteers were followed for 28 days, and gametocyte carriage was assessed. Direct skin feeding assays were performed on gametocyte carriers before and after ACT administration. RESULTS: Following artemether-lumefantrine treatment, gametocyte carriage decreased steadily from Day 0 to Day 21 post-treatment initiation. In contrast, for the artesunate-amodiaquine and artesunate-sulfadoxine/pyrimethamine arms, gametocyte carriage increased on Day 3 and remained constant until Day 7 before decreasing afterward. Mosquito feeding assays showed that artemether-lumefantrine and artesunate-amodiaquine significantly increased gametocyte infectivity to Anopheles gambiae sensu lato (s.l.) (p < 10-4), whereas artesunate-sulfadoxine/pyrimethamine decreased gametocyte infectivity in this setting (p = 0.03). CONCLUSION: Different ACT regimens could lead to gametocyte populations with different capacity to infect the Anopheles vector. Frequent assessment of the effect of antimalarials on gametocytogenesis and gametocyte infectivity may be required for the full assessment of treatment efficacy, the potential for spread of drug resistance and malaria transmission in the field.

O-fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii.

Toxoplasma gondii is an intracellular parasite that causes disseminated infections in fetuses and immunocompromised individuals. Although gene regulation is important for parasite differentiation and pathogenesis, little is known about protein organization in the nucleus. Here we show that the fucose-binding Aleuria aurantia lectin (AAL) binds to numerous punctate structures in the nuclei of tachyzoites, bradyzoites, and sporozoites but not oocysts. AAL also binds to Hammondia and Neospora nuclei but not to more distantly related apicomplexans. Analyses of the AAL-enriched fraction indicate that AAL binds O-linked fucose added to Ser/Thr residues present in or adjacent to Ser-rich domains (SRDs). Sixty-nine Ser-rich proteins were reproducibly enriched with AAL, including nucleoporins, mRNA-processing enzymes, and cell-signaling proteins. Two endogenous SRDs-containing proteins and an SRD-YFP fusion localize with AAL to the nuclear membrane. Superresolution microscopy showed that the majority of the AAL signal localizes in proximity to nuclear pore complexes. Host cells modify secreted proteins with O-fucose; here we describe the O-fucosylation pathway in the nucleocytosol of a eukaryote. Furthermore, these results suggest O-fucosylation is a mechanism by which proteins involved in gene expression accumulate near the NPC.

Dynamics of the Toxoplasma gondii inner membrane complex.

Unlike most cells, protozoa in the phylum Apicomplexa divide by a distinctive process in which multiple daughters are assembled within the mother (schizogony or endodyogeny), using scaffolding known as the inner membrane complex (IMC). The IMC underlies the plasma membrane during interphase, but new daughters develop in the cytoplasm, as cytoskeletal filaments associate with flattened membrane cisternae (alveolae), which elongate rapidly to encapsulate subcellular organelles. Newly assembled daughters acquire their plasma membrane as they emerge from the mother, leaving behind vestiges of the maternal cell. Although the maternal plasma membrane remains intact throughout this process, the maternal IMC disappears - is it degraded, or recycled to form the daughter IMC? Exploiting fluorescently tagged IMC markers, we have used live-cell imaging, fluorescence recovery after photobleaching (FRAP) and mEos2 photoactivation to monitor the dynamics of IMC biogenesis and turnover during the replication of Toxoplasma gondii tachyzoites. These studies reveal that the formation of the T. gondii IMC involves two distinct steps - de novo assembly during daughter IMC elongation within the mother cell, followed by recycling of maternal IMC membranes after the emergence of daughters from the mother cell.

Cryptic organelle homology in apicomplexan parasites: insights from evolutionary cell biology.

The economic and clinical significance of apicomplexan parasites drives interest in their many evolutionary novelties. Distinctive intracellular organelles play key roles in parasite motility, invasion, metabolism, and replication, and understanding their relationship with the organelles of better-studied eukaryotic systems suggests potential targets for therapeutic intervention. Recent work has demonstrated divergent aspects of canonical eukaryotic components in the Apicomplexa, including Golgi bodies and mitochondria. The apicoplast is a relict plastid of secondary endosymbiotic origin, harboring metabolic pathways distinct from those of host species. The inner membrane complex (IMC) is derived from the cortical alveoli defining the superphylum Alveolata, but in apicomplexans functions in parasite motility and replication. Micronemes and rhoptries are associated with establishment of the intracellular niche, and define the apical complex for which the phylum is named. Morphological, cell biological and molecular evidence strongly suggest that these organelles are derived from the endocytic pathway.

Toxoplasma gondii seroprevalence in Mali.

The protozoan parasite Toxoplasma gondii is globally distributed, with considerable local variation in prevalence based on behavioral and environmental factors. To assess prevalence and estimate risk in Mali, we conducted a survey of 760 serum samples previously collected for malaria studies. A modified agglutination test detected antibodies in ∼27% of the adult population, with no significant differences between men and women, or between urban and rural study sites. In the village of Kolle, seroprevalence rose from 0% in infants (<1 yr, but after weaning of maternal immunoglobulin G) to 0.8% (1-5 yr), 2.7% (6-10), 11.3% (11-15), and 26.8% (>15); differences between the <10-, 11-15-, and >15-yr age groups were highly significant (P ≤ 0.01). We also observed an increase in anti- T. gondii antibody titers with age. Modeling the observed age distribution suggests a seroconversion rate of ∼1%/yr, indicating that congenital toxoplasmosis may be an under-appreciated public health concern in Mali.