Falciparum Research Articles

Fc-Afucosylation of VAR2CSA-Specific Immunoglobulin G and Clinical Immunity to Placental Plasmodium falciparum Malaria.

Acquired immunity to Plasmodium falciparum malaria is mainly mediated by immunoglobulin G (IgG) targeting erythrocyte membrane protein 1 (PfEMP1). These adhesins mediate infected erythrocyte (IE) sequestration, protecting IEs from splenic destruction. PfEMP1-specific IgG is therefore thought to protect mainly by inhibiting IE sequestration. VAR2CSA-type PfEMP1 mediates placental IE sequestration, putting pregnant women exposed to P falciparum parasites at risk of placental malaria (PM). Levels and Fc-afucosylation of VAR2CSA-specific plasma IgG were measured by a modified enzyme-linked immunosorbent assay (FEASI). We also measured the ability of the IgG to inhibit IE adhesion and to induce natural killer (NK) cell degranulation. The results were related to parity and clinical pregnancy outcomes. Parity was positively correlated with levels and Fc-afucosylation of VAR2CSA-specific IgG, and with birth weight and plasma IgG inhibition of IE adhesion in vitro. Fc-afucosylation of VAR2CSA-specific IgG increased NK-cell degranulation. Women with Fc-afucosylated VAR2CSA-specific IgG had a reduced risk of delivering a low birth weight (LBW) baby, but not of PM or anemia. Fc-afucosylated VAR2CSA-specific IgG effectively induced NK-cell degranulation and was associated with protection against LBW, independent of IgG levels. Our study has implications for the development of VAR2CSA-based subunit vaccines, which exclusively induce Fc-fucosylated IgG.

Co-encapsulation of antimalarial drug and SPIO in glucose-conjugated polymeric micelles against parasite-infected erythrocytes

Malaria, a life-threatening infectious disease transmitted through bites of Anopheles mosquitoes, remains a major global health challenge. The erythrocytic stage of malaria, where the parasite undergoes multiplication within red blood cells, is crucial for disease progression. Targeting this stage is essential for effective treatment and eradication of the parasite. However, the emergence of drug resistance highlights the need for innovative therapeutic approaches. Furthermore, accurate detection and tracking of parasites are imperative for confirming complete parasite eradication. Enhancing drug efficacy and specifically targeting molecular entities are key strategies to address this challenge. To combat the disease, a drug delivery system was developed to encapsulate the drug along with contrast agents. This system was conjugated with glucose to target both new permeable pathways (NPPs) and the Plasmodium falciparum hexose transporter (PfHT). In this study, glucose-conjugated polymeric micelles containing superparamagnetic ironoxide (SPIO) nanoparticles and quinine were synthesized and characterized. These micelles demonstrated hemocompatibility and exhibited superior drug release profiles under acidic conditions mimicking the parasite's environment. In vitro studies on infected red blood cells (RBCs) revealed targeted micelles enhanced antimalarial activity compared to non-targeted micelles and free drug. Additionally, targeted micelles exhibited the highest fluorescence signal intensity and MRI contrast enhancement. Based on these results, glucose/quinine/SPIO micelles hold significant promise for tracking and treatment functions in malaria, offering precise detection and targeted drug delivery to eradicate the parasite effectively.

Socio-demographic factors, housing characteristics, and clinical symptoms associated with falciparum malaria in two rapidly urbanizing areas in the Ashanti region of Ghana

BackgroundMalaria has been described as a disease of poverty, affecting the poorest populations typically living in rural areas. As hitherto rural areas transition into semi-urban environments, this study investigated the prevalence of falciparum malaria and associated risk factors in two rapidly urbanizing districts in the Ashanti Region of Ghana.MethodsA cross-sectional, hospital-based study was conducted at Agona and Mankranso Government Hospitals located within the Sekyere South and Ahafo Ano Southwest districts respectively, in the Ashanti Region of Ghana. Five µL of venous blood was obtained from suspected malaria patients and tested for malaria using rapid diagnostic test (RDT). Data on socio-demographic factors, clinical symptoms, and housing characteristics were collected using a structured questionnaire. Univariate and multivariate logistic regression analysis were performed to identify risk factors associated with malaria.ResultsA total of 1739 participants were enrolled in the study between January and June 2021 with median age of 22 years (IQR = 6–36). Overall malaria prevalence was 24.8%. Compared to > 30-year-olds, children between 0 and 5 years (aOR = 3.36) and those aged between 6 and 14 (aOR = 6.71) were three and six times more likely to test positive for malaria, respectively. Similarly, farming (aOR = 1.74), compared to other occupations, living close to stagnant water (aOR = 1.34), experiencing chills (aOR = 1.5), and vomiting (aOR = 1.93) were associated with increased odds of malaria infection. Having roofing ceiling (aOR = 0.66) and screened doors (aOR = 0.75) were associated with decreased risk of malaria. However, sleeping under insecticide-treated nets (ITNs), using mosquito coils/repellents, and indoor residual spraying (IRS) were not statistically significantly associated with infection.ConclusionChildren between 0 and 5 years and those aged between 6 and 14 years continue to shoulder the highest burden of malaria. Efforts to improve housing characteristics such as installation of roofing ceiling, screening doors, and clearing potential mosquito breeding sites should be encouraged in these rapidly urbanizing areas.

Safety and Efficacy of Immunization with a Late-Liver-Stage Attenuated Malaria Parasite

BackgroundCurrently licensed and approved malaria subunit vaccines provide modest, short-lived protection against malaria. Immunization with live-attenuated Plasmodium falciparum malaria parasites is an alternative vaccination strategy that has potential to improve protection.MethodsWe conducted a double-blind, controlled clinical trial to evaluate the safety, side-effect profile, and efficacy of immunization, by means of mosquito bites, with a second-generation genetically attenuated parasite (GA2) — a mei2 single knockout P. falciparum NF54 parasite (sporozoite form) with extended development into the liver stage. After an open-label dose-escalation safety phase in which participants were exposed to the bites of 15 or 50 infected mosquitoes (stage A), healthy adults who had not had malaria were randomly assigned to be exposed to 50 mosquito bites per immunization of GA2, an early-arresting parasite (GA1), or placebo (bites from uninfected mosquitoes) (stage B). After the completion of three immunization sessions with 50 mosquito bites per session, we compared the protective efficacy of GA2 against homologous P. falciparum controlled human malaria infection with that of GA1 and placebo. The primary end points were the number and severity of adverse events (in stages A and B) and blood-stage parasitemia greater than 100 P. falciparum parasites per milliliter after bites from GA2-infected mosquitoes (in stage A) and after controlled human malaria infection (in stage B).ResultsAdverse events were similar across the trial groups. Protective efficacy against subsequent controlled human malaria infection was observed in 8 of 9 participants (89%) in the GA2 group, in 1 of 8 participants (13%) in the GA1 group, and in 0 of 3 participants in the placebo group. A significantly higher frequency of P. falciparum–specific polyfunctional CD4+ and Vδ2+ γδ T cells were observed among participants who received GA2 than among those who received GA1, whereas GA2 and GA1 induced similar antibody titers targeting the P. falciparum circumsporozoite protein.ConclusionsIn this small trial, GA2 was associated with a favorable immune induction profile and protective efficacy, findings that warrant further evaluation. (Funded by the Bontius Foundation; ClinicalTrials.gov number, NCT04577066.)

Genotyping and Characterizing Plasmodium falciparum to Reveal Genetic Diversity and Multiplicity of Infection by Merozoite Surface Proteins 1 and 2 (msp-1 and msp-2) and Glutamate-Rich Protein (glurp) Genes.

Resistance to current antimalarial drugs is steadily increasing, and new drugs are required. Drug efficacy trials remain the gold standard to assess the effectiveness of a given drug. The World Health Organization (WHO)'s recommendation for the optimal duration of follow-up for assessing antimalarial efficacy is a minimum of 28 days. However, assessing antimalarial drug efficacy in highly endemic regions can be challenging due to the potential risks of acquiring a new infection in the follow-up period, and thus, it may underestimate the efficacy of the given drugs. A new treatment should be introduced if treatment failure rates exceed 10%. Overestimation occurs as a result of retaining a drug with a clinical efficacy of less than 90% with increases in morbidity and mortality, while underestimation may occur due to a misclassification of new infections as treatment failures with tremendous clinical and economic implications. Therefore, molecular genotyping is necessary to distinguish true new infections from treatment failures to ensure accuracy in determining antimalarial efficacy. There are three genetic markers that are commonly used in antimalarial efficiency trials to discriminate between treatment failures and new infections. These include merozoite surface protein 1 (msp-1), merozoite surface protein 2 (msp-2), and glutamate-rich protein (glurp). The genotyping of P. falciparum by nested polymerase chain reaction (n-PCR) targeting these markers is discussed with the inherent limitations and uncertainties associated with the PCR technique and limitations enforced by the parasite's biology itself.

Fatal Mixed Plasmodium Infection in Traveler Returning to Colombia from Comoros Islands, 2024.

During 2014-2022, only Plasmodium falciparum malaria cases were reported in the Comoro Islands. We report a fatal case of mixed Plasmodium malaria infection in a traveler returning from the Comoros to Colombia in 2024, highlighting the need to strengthen laboratory detection and identification of Plasmodium spp. in sub-Saharan Africa.

Antiplasmodial potential of compounds isolated from Ziziphus mucronata and their binding to Plasmodium falciparum HGXPRT using biophysical and molecular docking studies.

The increasing resistance of Plasmodium parasites to currently available antiplasmodial therapies poses a significant challenge in treating malaria. Since ancient times, plants have served as a primary source of novel pharmacologically active compounds for drug development. Therefore, this study aimed to explore the antiplasmodial properties of pentacyclic triterpenes isolated from Ziziphus mucronata bark, with an emphasis on their mechanism of action. Dichloromethane and ethyl acetate extracts of the stem bark were subjected to silica gel column chromatography, which led to the isolation of three known triterpenoids: betulinic acid, methyl betulinate, and lupeol. The compounds were then evaluated for antiplasmodial activity against Plasmodium falciparum NF54 strains using the Plasmodium lactate dehydrogenase (pLDH) assay. In silico evaluation of the isolated compounds was conducted through molecular docking and further validated with in vitro experiments against a purified protein target, Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT). Betulinic acid, methyl betulinate, and lupeol exhibited potent antiplasmodial activities with IC50 values of 20, 10.11, and 7.56µg/mL, respectively. Lupeol exhibited the highest binding energy of - 7.6kcal/mol. Differential scanning fluorimetry revealed that lupeol decreases the Tm of PfHGXPRT, thus decreasing the protein's thermal stability. At high concentrations, lupeol also increased protein absorbance, indicating the detection of hydrophobic amino acids and protein unfolding. This study proves that Z. mucronata could serve as a reservoir of effective agents for treating malaria, while also scientifically validating its use in traditional medicine. However, further experimental studies are required to substantiate its relevant therapeutic effects.

Identification of antimalarial phytoconstituents from Tinospora sinensis (Lour.) Merr. Stem by in vitro whole cell assay and multiple targets directed in silico screening against Plasmodium falciparum

Ethnopharmacological relevanceTinospora sinensis (Lour.) Merr., from the family Menispermaceae, is widely used in Indian folk and Ayurvedic medicine. Indigenous tribes such as the Tea-tribe and Chorei-tribe of Assam use its bark and stem as a herbal remedy to treat malaria and it is also traditionally employed for conditions such as dyspepsia, inflammation, fever, ulcers, jaundice, diabetes and various urinary, skin, and liver diseases. Aim of the studyThis study aims to identify and characterize antimalarial phytoconstituents from the active extract of T. sinensis stem by in vitro screening against both the Chloroquine-sensitive (Pf3D7) as well as Chloroquine-resistant (PfRKL-9) strains of Plasmodium falciparum, along with exploring potential targets and mechanisms using molecular docking and dynamics simulation studies. Materials and methodsT. sinensis stems were collected from Assam, India, and authenticated by the Botanical Survey of India. The plant materials were initially extracted with non-polar to polar solvents and screened for in vitro antimalarial potency against Pf3D7 and PfRKL-9. Then, the methanol extract was selected for bioassay-guided isolation of phytoconstituent(s). The isolated phytoconstituent(s) were screened for antimalarial potential and active compounds were further evaluated for cytotoxicity using the HEK-293 cell line. Structural characterization of the active compounds involved the use of UV–VIS, IR, NMR and HRMS analyses. Molecular docking and dynamics simulation studies were performed on selected targets from P. falciparum to predict binding affinities and mechanisms of action. ResultsFrom the methanol extract of T. sinensis stem, five phytoconstituents were isolated, including isoquinoline alkaloids Berberine (NG1) and Palmatine (NG2) showed the best antimalarial activity (IC50 < 1 μg/ml) against both Pf3D7 and PfRKL-9. Cytotoxicity assays confirmed their safety and selectivity. Molecular docking and dynamic simulation studies revealed that Berberine and Palmatine formed stable complexes with P. falciparum lysyl-tRNA synthetase and P. falciparum aminopeptidase N, respectively, indicating their potential as antimalarial leads. ConclusionThis study identifies two potent antimalarial phytoconstituents in the stem of T. sinensis, validating its traditional use and demonstrating its safety and efficacy for potential global application in malaria treatment.

Transcription and Copy Number Variation of Plasmodium falciparum var2csa among Nonpregnant Malaria Patients in Thailand.

Placental malaria is an important cause of fetomaternal morbidity and mortality among pregnant women infected with Plasmodium falciparum. The pathogenesis involves the binding of VAR2CSA on the surface of infected erythrocytes to chondroitin sulfate proteoglycans on syncytiotrophoblasts in the intervillous space of the placenta. Anti-VAR2CSA antibodies confer protection from adverse pregnancy outcomes in falciparum malaria; therefore, VAR2CSA is a strong vaccine candidate against placental malaria. To date, little is known about transcription of var2csa among isolates from male and nonpregnant patients in low transmission areas. In this study, transcription and copy number variation of var2csa were analyzed in 55 P. falciparum isolates from nonpregnant women, men and children with symptomatic malaria in five endemic provinces of Thailand. Reverse transcription polymerase chain reaction (PCR) detected var2csa transcripts in 43 (78.2%) blood samples. Multiple copies of var2csa were identified in 16 of 55 (29.1%) isolates by quantitative real-time PCR. Copy number variation of var2csa was not associated with the patients' gender, age group, ethnicity, parasite genotypes, parasite density, and geographic origins. These data suggest that P. falciparum carrying multicopy var2csa had a wide geographic distribution in Thailand with a prevalence rate comparable to those observed in high transmission areas of Africa. The high prevalence of isolates from male and nonpregnant patients with var2csa transcription suggests that the transcription of this gene occurs naturally during blood stage infection. Whether VAR2CSA is expressed and immunogenic among nonpregnant falciparum malaria patients requires further study.

Severe Malaria in Angola: The Clinical Profile and Disease Outcome Among Adults from a Low-Endemic Area.

Severe malaria poses a significant public health concern in Angola, particularly among adults. This study assessed the clinical manifestations and outcomes of severe Plasmodium falciparum malaria in adult patients admitted to Hospital Central Dr. António Agostinho Neto of Lubango (HCL), Angola. The study retrospectively reviewed medical records of patients over 14 years old admitted with severe malaria during the first quarter of 2021 and 2022, coinciding with the peak transmission season. The World Health Organization (WHO) criteria were used to clarify the disease severity. The cohort included 640 patients-167 in 2021 and 473 in 2022-distributed across the following departments: the Intensive Care Unit (ICU; n = 81), Medicine (MED; n = 458) and Infectiology (INF; n = 101). The median age was 26 years and 59.4% were males. Renal impairment was the most frequent severe manifestation, affecting 37.4% of cases. The mortality rate across the study period was 7%, showing a notable decrease from 10.2% in 2021 to 5.9% in 2022. The higher mortality rate in 2021 may reflect the impact of the COVID-19 pandemic, which limited hospital access and delayed care, resulting in more critical cases being admitted at a later stage. In 2022, with reduced COVID-19 pressures, earlier access to treatment may have improved outcomes, contributing to the lower mortality rate. This study emphasizes the need to assess the clinical burden of severe malaria in low-endemic regions, where shifting patterns may signal emerging threats such as antimalarial drug resistance. Further research is essential to optimize control strategies and strengthen surveillance systems, reducing morbidity and mortality.

Accelerating Antimalarial Drug Discovery with a New High-Throughput Screen for Fast-Killing Compounds.

The urgent need for rapidly acting compounds in the development of antimalarial drugs underscores the significance of such compounds in overcoming resistance issues and improving patient adherence to antimalarial treatments. The present study introduces a high-throughput screening (HTS) approach using 1536-well plates, employing Plasmodium falciparum lactate dehydrogenase (PfLDH) combined with nitroreductase (NTR) and fluorescent probes to evaluate inhibition of the growth of the asexual blood stage of malaria parasites. This method was adapted to efficiently assess the speed of action profiling (SAP) in a 384-well plate format, streamlining the traditionally time-consuming screening process. By successfully screening numerous compounds, this approach identified fast-killing hits early in the screening process, addressing challenges associated with artemisinin-based combination therapies. The high-throughput SAP method is expected to be of value in continuously monitoring fast-killing properties during structure-activity relationship studies, expediting the identification and development of novel, rapidly acting antimalarial drugs within phenotypic drug discovery campaigns.

Estimation of PfRh5-based vaccine efficacy in asymptomatic Plasmodium falciparum patients from high-endemic areas of Tanzania using genetic and antigenicity variation screening.

Plasmodium falciparum is the most lethal malaria parasite. Recent phase 1b vaccine trials using P. falciparum reticulocyte binding protein homolog 5 (PfRh5) demonstrated safety and promising efficacy in preventing merozoite invasion. PfRh5 has emerged as a strong vaccine candidate due to its essential role in merozoite invasion and limited sequence variation. For effective malaria vaccine development, especially in high-transmission settings, strain-transcending activity must be considered. Ongoing monitoring of antigenic variation and natural immune responses is important to estimate vaccine efficacy across geographically diverse populations. Samples for this study were collected from four villages in each of the Kigoma and Geita regions, known malaria transmission hotspots in Tanzania. This community-based cross-sectional study was conducted from December 2022 to July 2023. Genetic variation and natural selection pressure on pfrh5 were analyzed in 164 asymptomatic P. falciparum isolates. The humoral immune response to PfRh5 was also assessed using a protein microarray with 242 sera samples from asymptomatic patients in the same population. Finally, a correlation analysis was conducted to compare pfrh5 genetic variation with the humoral immune response. The results revealed that pfrh5 was well conserved, but novel non-synonymous mutations were found at D65H, H170N, and I227M. Additionally, natural selection metrics indicated the potential for positive selection and a recent population expansion of PfRh5 in the study area, both of which could influence vaccine effectiveness. Antigenicity screening revealed variable sensitivity, ranging from 3.3% in Bunyambo to 82.8% in Rwantaba, with no significant relationship between antigenicity and parasitemia, haplotypes, or gender. However, age was significantly associated with humoral immune response (ρ = 0.170, p = 0.008). These findings underscore the need for future PfRh5-based vaccines to consider for increasing genetic variation and geographical differences in humoral immune responses.

Probing novel epitopes on the Plasmodium falciparum circumsporozoite protein for vaccine development

RTS,S and R21 are the only vaccines recommended by the WHO to protect children from Plasmodium falciparum (Pf) clinical malaria. Both vaccines target the Pf sporozoite surface protein circumsporozoite protein (CSP). Recent studies showed that human antibodies neutralize Pf sporozoites most efficiently when simultaneously binding to the PfCSP NANP repeat and the NPDP junction domain. However, neither RTS,S nor R21 targets this junction domain. To test the potential of the NPDP junction domain and other sites of PfCSP as innovative vaccine targets, we developed multiple vaccine candidates based on cucumber mosaic virus-like particles (CuMVTT-VLPs). These candidates vary in several aspects: the number of targeted NANP repeats, the presence or absence of the junction domain, the cleavage site, and up to three NVDP repeats within the target sequence. Immunogenicity and efficacy studies were conducted in BALB/c mice, utilizing chimeric Plasmodium berghei (Pb) sporozoites, in which the endogenous CSP has been replaced by PfCSP (Pb/PfCSP). We observed a positive association between the number of targeted NANP repeats and the induction of specific IgM/IgG antibodies. Elevated humoral responses led to enhanced protection against parasitemia after Pb/PfCSP sporozoite challenge. Especially high-avidity/affinity antibody formation and vaccine protection were NANP repeat-dependent. Intriguingly, vaccine efficacy was not enhanced by targeting sites on PfCSP other than the NANP repeats. Our data emphasize the dominant role of the NANP repeat region for induction of protective antibodies. Furthermore, we present here novel malaria vaccine candidates with an excellent immunogenic profile that confer sterile protection in mice, even in absence of adjuvants.

Kidney involvement in Plasmodium falciparum infection in a pregnant patient

BackgroundThe course of kidney function and outcomes of severe malaria infection in pregnant women is poorly understood. The indications for renal replacement therapy in pregnant patients with AKI are similar to the general population. This is the case of a pregnant patient with severe Plasmodium falciparum infection that caused cerebral malaria, acute kidney injury (AKI) who required renal replacement therapy and kidney biopsy during her hospitalization.Case presentationA 29-year-old pregnant woman from Equatorial Guinea was admitted to the hospital with haemolytic anaemia, hyperbilirubinaemia and thrombocytopenia. During hospitalization, a thick blood smear was performed where parasitaemia by P. falciparum were observed and confirmed by real-time PCR assay. The patient developed cerebral malaria secondary to an ischaemic-type cerebral vascular event, hypotension and severe. After confirming diagnosis of P. falciparum infection, artesunate, artemether/lumefantrine and primaquine were started. Kidney biopsy revealed an active tubulointerstitial nephritis with acute tubular lesion and pigment tubulopathy with negative immunofluorescence. After CVVHDF, the patient received intermittent haemodialysis until the recovery of kidney function. After discharge, follow-up was carried until the successful resolution of the pregnancy by cesarean delivery and not shown deterioration in kidney function or proteinuria.ConclusionIn this case, intensive dialysis was started and dialysis intensity progressively reduced when kidney function improved. Due to the evolution of kidney function, a kidney biopsy was performed which showed tubulointerstitial nephritis as a manifestation of the infection. While the kidney biopsy was of interest for discriminating between tubular and glomerular involvement, the availability of placental biomarkers (sflt1-PlGF) would have been of help for ruling out preeclampsia and placental damage. The multidisciplinary approach to AKI during pregnancy should be the rule, with diligent care of maternal–fetal well-being during pregnancy and monitoring of kidney function after delivery.

The J Domain Proteins of Plasmodium knowlesi, a Zoonotic Malaria Parasite of Humans.

Plasmodium knowlesi is a zoonotic form of human malaria, the pathology of which is poorly understood. While the J domain protein (JDP) family has been extensively studied in Plasmodium falciparum, and shown to contribute to malaria pathology, there is currently very limited information on the P. knowlesi JDPs (PkJDPs). This review provides a critical analysis of the literature and publicly available data on PkJDPs. Interestingly, the P. knowlesi genome encodes at least 31 PkJDPs, with well over half belonging to the most diverse types which contain only the signature J domain (type IIIs, 19) or a corrupted version of the J domain (type IVs, 2) as evidence of their membership. The more typical PkJDPs containing other domains typical of JDPs in addition to the J domain are much fewer in number (type IIs, 8; type Is, 2). This study indentifies PkJDPs that are potentially involved in: folding of newly synthesized or misfolded proteins within the P. knowlesi cytosol (a canonical type I and certain typical type IIs); protein translocation (a type III) and folding (a type II) in the ER; and protein import into mitochondria (a type III). Interestingly, a type II PkJDP is potentially exported to the host cell cytosol where it may recruit human HSP70 for the trafficking and folding of other exported P. knowlesi proteins. Experimental studies are required on this fascinating family of proteins, not only to validate their role in the pathology of knowlesi malaria, but also because they represent potential anti-malarial drug targets.

Severity of Vessel Color Changes and Macular and Peripheral Whitening in Malarial Retinopathy Are Associated with Higher Total Body and Sequestered Parasite Burdens.

Two-thirds of children with cerebral malaria (CM) exhibit retinopathy characterized by whitening, vessel color changes, and/or hemorrhages. The pathogenesis of malarial retinopathy is not fully understood. This study aimed to assess the relationship between malarial retinopathy and the severity of its components (macular whitening, retinal hemorrhages, and vessel color changes) with the total, circulating, or sequestered parasite load in children with CM. Total parasite burden was estimated by measuring plasma levels of Plasmodium falciparum histidine-rich protein 2 (PfHRP2), while the sequestered load was calculated as the difference between the total burden and circulating parasitemia. Children with retinopathy-positive CM (n = 172) had higher total and sequestered parasite burdens compared to retinopathy-negative children (n = 42) (both p = 0.049). In a subgroup with detailed retinopathy grading (n = 52), more extensive vessel color changes correlated with higher total, sequestered, and circulating parasite loads (p = 0.0057, p = 0.0068, and p = 0.0433, respectively). Peripheral retinal whitening was also associated with increased total and sequestered loads (p = 0.0017 and p = 0.0012). No association was found between retinal hemorrhages and parasite burden, indicating that other factors may influence their pathogenesis.

The extended recovery ring-stage survival assay is a scalable alternative for artemisinin susceptibility phenotyping of fresh Plasmodium falciparum isolates.

Artemisinin partial resistance (ART-R) has emerged in eastern Africa, necessitating regular surveillance of susceptibility of Plasmodium falciparum to artemisinins. The microscopy-based ring-stage survival assay (RSA) provides a laboratory correlate of ART-R but is limited by low throughput and subjectivity of microscopic counts of viable parasites. The extended recovery ring-stage survival assay (eRRSA) replaces microscopy with efficient quantitative PCR (qPCR) readouts but has been studied only with culture-adapted P. falciparum clones. We measured susceptibility to dihydroartemisinin (DHA) after a 6-h incubation with 700-nM DHA, followed by culture without drug, by comparing survival with that of untreated controls by microscopy (the RSA) or qPCR (the eRRSA) and also performed standard growth inhibition (half-maximal inhibitory concentration [IC50]) assays for 122 P. falciparum isolates freshly collected in eastern and northern Uganda from March to July 2022. The median values for RSA survival, eRRSA fold change, and DHA IC50 were 3.0%, 46.2, and 3.2 nM, respectively. RSA percent survival and eRRSA fold changes correlated strongly (Spearman correlation coefficient [rs] = -0.7411, P < 0.0001), with modest associations between the presence of validated P. falciparum Kelch13 ART-R mutations (C469Y or A675V) and RSA (median survival 2.6% for wild type [WT] vs 4.1% for mutant, P = 0.01), or eRRSA (median fold change 63.4 for WT vs 30.9 for mutant, P = 0.003) results. Significant correlations were also observed between DHA IC50 values and both RSA percent survival (rs = 0.4235, P < 0.0001) and eRRSA fold changes (rs = -0.4116, P < 0.0001). The eRRSA is a scalable alternative for phenotyping fresh P. falciparum isolates, providing similar results with improved throughput.

Efficacy of attractive targeted sugar bait stations against malaria in Western Province Zambia: epidemiological findings from a two-arm cluster randomized phase III trial

BackgroundAttractive targeted sugar bait (ATSB) stations containing bait (to attract) and ingestion toxicant (to kill) sugar-foraging mosquitoes are hypothesized to reduce malaria transmission by shortening the lifespan of Anopheles vectors.MethodsA two-arm cluster-randomized controlled trial (cRCT) was conducted in Western Province Zambia. Seventy clusters of 250–350 households were assigned (1:1) by restricted randomization to an intervention arm (ATSB) or control arm (no ATSB) in the context of standard of care vector control (insecticide-treated nets and/or indoor residual spraying). Two ATSB stations (Westham Sarabi, 0.11% dinotefuran w/w) were maintained on exterior walls of eligible household structures for a 7-month deployment period (December-June) during the high malaria transmission season. The primary outcome was clinical malaria incidence among two consecutive seasonal cohorts of children aged 1–14 years, followed-up monthly from January-June in 2022 and 2023. Secondary outcome was Plasmodium falciparum prevalence among individuals aged over six months. Analysis compared clinical malaria incidence and prevalence between arms among the intention-to-treat population.ResultsATSB coverage, assessed by cross-sectional survey, was 98.3% in March–April 2022 and 89.5% in March–April 2023. 4494 children contributed any follow-up time to the cohort, with 2313 incident malaria cases in the intervention arm (1.28 per child per six-month transmission season), and 2449 in the control arm (1.38 per child-season). The incidence rate ratio between the two arms was 0.91 (95% CI 0.72–1.15, p = 0.42). 2536 individuals participated in cross-sectional surveys, with prevalence of P. falciparum 50.7% in the intervention arm and 53.5% in the control arm. The odds ratio between the two arms was 0.89 (95% CI 0.66–1.18, p = 0.42). Secondary covariable-adjusted and subgroup analyses did not substantially alter the findings. No serious adverse events associated with the intervention were reported.ConclusionsTwo ATSB stations deployed per eligible structure for two consecutive transmission seasons did not result in a statistically significant reduction in clinical malaria incidence among children aged 1–14 years or in P. falciparum prevalence in rural western Zambia. Further studies are needed to assess the efficacy of ATSB stations in different settings and with different deployment strategies.Trial registrationThe trial is registered with Clinicaltrials.gov (NCT04800055).

Polymorphisms in the Pfcrt, Pfmdr1, and Pfk13 genes of Plasmodium falciparum isolates from southern Brazzaville, Republic of Congo

This study aimed to analyze polymorphisms in Pfcrt, Pfmdr1, and Pfk13 genes’ markers of resistance to Artemisinin-based combination therapy (ACT), in Plasmodium falciparum isolates from southern Brazzaville, 15 years after the adoption of ACT in the Republic of Congo. A total of 369 microscopy-confirmed malaria-infected individuals were enrolled from March to October 2021 in the community and in health facilities during a cross-sectional study. The K76T mutation in the Pfcrt gene, N86Y and Y184F mutations in the Pfmdr1 gene were investigated using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) while the codons region (1005–1300) of the Pfmdr1gene, and Pfk13 gene were sequenced. The prevalences of K76T, N86Y, Y184F mutations were 26.0%, 6.8%, and 27.7%, respectively. However, no mutations were detected in codons 1034, 1042, and 1246 of the Pfmdr1 gene. None of the mutations previously associated with artemisinin-based resistance were detected in the Pfk13 gene. The results reveal a significant decrease in the prevalence of K76T, N86Y, Y184F mutations, in Plasmodium falciparum isolates following the change of therapeutic policy. As artemisinin resistance is emerging throughout Africa, continued surveillance for early detection of these mutations and relevant partner markers of drug resistance are recommended in the Republic of Congo.

Plasmodium falciparum genetic diversity and multiplicity of infection among asymptomatic and symptomatic malaria-infected individuals in Uganda

BackgroundPlasmodium falciparum (P. falciparum) remains a significant public health challenge globally, especially in sub-Saharan Africa (SSA), where it accounts for 99% of all malaria infections. The outcomes of P. falciparum infection vary, ranging from asymptomatic to severe, and are associated with factors such as host immunity, parasite genetic diversity, and multiplicity of infection (MOI). Using seven neutral microsatellite markers, the current study investigated P. falciparum genetic diversity and MOI in both asymptomatic and symptomatic malaria individuals in Uganda.MethodsThis cross-sectional study analyzed 225 P. falciparum isolates from both asymptomatic and symptomatic malaria patients, ranging in age from 6 months to ≥ 18 years. P. falciparum genetic diversity, MOI, and multi-locus linkage disequilibrium (LD) were assessed through genotyping of seven neutral microsatellite markers: Poly-α, TA1, TA109, PfPK2, 2490, C2M34–313, and C3M69–383. Genetic data analysis was performed using appropriate genetic analysis software.ResultsP. falciparum infections exhibited high genetic diversity in both asymptomatic and symptomatic individuals. The mean expected heterozygosity (He) ranged from 0.79 in symptomatic uncomplicated malaria cases to 0.81 in asymptomatic individuals. There was no significant difference (p = 0.33) in MOI between individuals with asymptomatic and symptomatic infections, with the mean MOI ranging from 1.92 in symptomatic complicated cases to 2.10 in asymptomatic individuals. Polyclonal infections were prevalent, varying from 58.5% in symptomatic complicated malaria to 63% in symptomatic uncomplicated malaria cases. A significant linkage disequilibrium (LD) was observed between asymptomatic and symptomatic uncomplicated/complicated infections (p < 0.01). Genetic differentiation was low, with FST values ranging from 0.0034 to 0.0105 among P. falciparum parasite populations in asymptomatic and symptomatic uncomplicated/complicated infections.ConclusionThere is a high level of P. falciparum genetic diversity and MOI among both symptomatic and asymptomatic individuals in Uganda. Asymptomatic carriers harbor a diverse range of parasites, which poses challenges for malaria control and necessitates targeted interventions to develop effective strategies.