Malaria Journal

Anopheles funestus is playing an increasing role in malaria transmission in parts of sub-Saharan Africa, where An. gambiae s.s. has been effectively controlled by long-lasting insecticidal nets. We investigated vector population bionomics, insecticide resistance and malaria transmission dynamics in 86 study clusters in North-West Tanzania. An. funestus s.l. represented 94.5% (4740/5016) of all vectors and was responsible for the majority of malaria transmission (96.5%), with a sporozoite rate of 3.4% and average monthly entomological inoculation rate (EIR) of 4.57 per house. Micro-geographical heterogeneity in species composition, abundance and transmission was observed across the study district in relation to key ecological differences between northern and southern clusters, with significantly higher densities, proportions and EIR of An. funestus s.l. collected from the south. An. gambiae s.l. (5.5%) density, principally An. arabiensis (81.1%) and An. gambiae s.s. (18.9%), was much lower and closely correlated with seasonal rainfall. Both An. funestus s.l. and An. gambiae s.l. were similarly resistant to alpha-cypermethrin and permethrin. Overexpression of CYP9K1, CYP6P3, CYP6P4 and CYP6M2 and high L1014F-kdr mutation frequency were detected in An. gambiae s.s. populations. Study findings highlight the urgent need for novel vector control tools to tackle persistent malaria transmission in the Lake Region of Tanzania.

BackgroundTracking and understanding artemisinin resistance is key for preventing global setbacks in malaria eradication efforts. The ring-stage survival assay (RSA) is the current gold standard for in vitro artemisinin resistance phenotyping. However, the RSA has several drawbacks: it is relatively low throughput, has high variance due to microscopy readout, and correlates poorly with the current benchmark for in vivo resistance, patient clearance half-life post-artemisinin treatment. Here a modified RSA is presented, the extended Recovery Ring-stage Survival Assay (eRRSA), using 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives, including parasite isolates with and without kelch13 mutations. MethodsP. falciparum cultures were synchronized with single layer Percoll during the schizont stage of the erythrocytic cycle. Cultures were left to reinvade to early ring-stage and parasitemia was quantified using flow cytometry. Cultures were diluted to 2% hematocrit and 0.5% parasitemia in a 96-well plate to start the assay, allowing for increased throughput and decreased variability between biological replicates. Parasites were treated with 700nM of dihydroartemisinin or an equivalent amount of dimethyl sulfoxide (DMSO) for 6 h, washed three times in drug-free media, and incubated for 66 or 114 h, when samples were collected and frozen for PCR amplification. A SYBR Green-based quantitative PCR method was used to quantify the fold-change between treated and untreated samples. Results15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives were assayed using the eRRSA. Due to the large number of pyknotic and dying parasites at 66 h post-exposure (72 h sample), parasites were grown for an additional cell cycle (114 h post-exposure, 120 h sample), which drastically improved correlation with patient clearance half-life compared to the 66 h post-exposure sample. A Spearman correlation of 0.8393 between fold change and patient clearance half-life was identified in these 15 isolates from Southeast Asia, which is the strongest correlation reported to date. ConclusionseRRSA drastically increases the efficiency and accuracy of in vitro artemisinin resistance phenotyping compared to the traditional RSA, which paves the way for extensive in vitro phenotyping of hundreds of artemisinin resistant parasites.

BackgroundScreening for Plasmodium spp. sporozoite infection in mosquitoes is routinely done using ELISA (enzyme-linked immunosorbent assay). Near infrared spectroscopy (NIRS), a fast and non-destructive method, has recently been shown to distinguish, with 95% accuracy, between uninfected and sporozoite-infected mosquitoes using laboratory strains of Plasmodium falciparum (PfN54). The aim of this present study was to further investigate the reproducibility of NIRS to identify sporozoite infection in mosquitoes infected using field isolates of P. falciparum gametocytes from asymptomatic carriers. MethodsHealthy individuals (aged 5 years and above) were screened for gametocytaemia by thick-smear microscopy in an area of moderate transmission along the Coast of Kenya between May and September 2018. Asymptomatic gametocyte carriers were recruited for mosquito feeding assays, direct membrane feeding (DMFA) and direct skin feeding (DFA), using insectary-reared Anopheles gambiae s.s (Kilifi strain). Mosquitoes were kept for 14-days post feeding after which they were scanned using NIRS and subsequently analysed for sporozoite infection using circumsporozoite-ELISA. Predictive models were explored using partial least square regressions (PLS). ResultsTwo hundred and ninety-nine (299) individuals were screened for malaria parasites, 74 (24.8%) were found with circulating asexual parasites, and 16 (5.4%) with P. falciparum gametocyte stages. Fourteen (14) asymptomatic gametocyte carriers were recruited to the study for mosquito feeding assays. A total of 134 (7%, 134/1881) sporozoite-infected mosquitoes were obtained from 9 successful experiments. Three different training datasets composed of infected and uninfected mosquitoes were analysed. The PLS models were unable to distinguish between sporozoite-infected and uninfected mosquitoes. A predictive model could not be generated. ConclusionsThe results of this study were not consistent with previous published research on NIRS for detection of sporozoite infection in the same mosquito species and may reflect differences between laboratory and field conditions. The current findings indicate that methods for sporozoite detection should be tested on field isolates at an early stage in their development and are informative for future research seeking novel high-throughput methods for parasite detection in mosquitoes.

Malaria remains a major global health burden, with traditional control methods facing challenges such as insecticide resistance and high operational costs. Genetic biocontrol offers a promising alternative for mosquito population suppression, but its field efficacy would require assessment. This study evaluates the role that population genomic statistics can play in detecting decreases in population size in the context of a cluster randomized control trial (cRCT), investigating the response of nucleotide diversity ({pi}), Tajimas D, segregating sites, and linkage disequilibrium (LD) under both constant and seasonal demographic scenarios. We simulated 90% and 99% population declines with various degrees of between-cluster heterogeneity, and assessed the detection power of each statistic over time and number of clusters per arm. Results show that Tajimas D is highly sensitive and robust across crash severity, seasonality and heterogeneity scenarios. Segregating sites has similar power to Tajimas D when baseline data are available. We further estimated that cRCTs require approximately 3 to 5 villages per treatment arm to achieve adequate statistical power. These findings provide recommendations for genetic monitoring of vector control interventions in wild populations.

Pyrethroid-chlorfenapyr nets have demonstrated improved entomological and epidemiological impact in trials across Africa. This is driving increased demand for this novel net class in malaria endemic countries. PermaNet(R) Dual is a new deltamethrin-chlorfenapyr net developed by Vestergaard Sarl to provide more options to malaria control programmes. We performed an experimental hut trial to evaluate the efficacy of PermaNet(R) Dual against wild, free-flying pyrethroid-resistant Anopheles gambiae sensu lato in Cove, Benin. PermaNet(R) Dual induced superior levels of mosquito mortality compared to a pyrethroid-only net and a pyrethroid-piperonyl butoxide net both when unwashed (77% with PermaNet(R) Dual vs. 23% with PermaNet(R) 2.0 and 56% with PermaNet(R) 3.0, p<0.001) and after 20 standardised washes (75% with PermaNet(R) Dual vs. 14% with PermaNet(R) 2.0 and 30% with PermaNet(R) 3.0, p<0.001). Using a provisional non-inferiority margin defined by the World Health Organisation, PermaNet(R) Dual was also non-inferior to a pyrethroid-chlorfenapyr net that has demonstrated improved public health value (Interceptor(R) G2), for vector mortality (79% vs. 76%, OR=0.854, 95% CIs: 0.703-1.038) but not for blood-feeding protection (35% vs. 26%, OR=1.445, 95% CIs: 1.203-1.735). PermaNet(R) Dual presents an additional option of this highly effective net class for improved control of malaria transmitted by pyrethroid-resistant mosquitoes.

In the fight against malaria, transmission blocking interventions (TBIs) are promising approaches to complement conventional tools. They aim to prevent the infection of the vectors and thereby reduce the subsequent exposure of a human population to infectious mosquitoes. The effectiveness of these approaches has been shown to depend on the initial intensity of infection in mosquitoes, often measured as the mean number of oocysts resulting from an infectious blood meal prior to intervention. In mosquitoes exposed to a high infection load, current TBI candidates are expected to be ineffective at completely blocking infection but will decrease parasite load and therefore, potentially also affect key parameters of vector transmission. The present study aims to investigate the consequences of changes in oocyst intensity on downstream parasite development and mosquito survival. To address this, we experimentally produced different infection intensities in Anopheles gambiae females by diluting gametocytes from natural Plasmodium falciparum isolates and used a newly developed non-destructive method based on the exploitation of mosquito sugar feeding to track parasite and mosquito life history traits throughout sporogonic development. Our results indicate that P. falciparum extrinsic incubation period (EIP) and mosquito survival did not vary with parasite density but differed significantly between parasite isolates. Our results do not identify here unintended consequences of the decrease of parasite loads in mosquitoes on the parasite incubation period or on mosquito survival, two key parameters of vectorial capacity, and hence support the transmission blocking strategies to control malaria. Author summaryIn the fight against malaria, it is recognized that the use of several complementary strategies is necessary to significantly reduce transmission and improve human health. Among these, transmission blocking strategies aim at blocking the development of the parasites within the mosquito vectors. This approach should prevent infection in most of mosquitoes feeding on infectious hosts and thus block the transmission. However, in some cases it may only reduce parasite load without fully clearing the infection. Here we identified potential risks: if reducing parasite load would reduce the incubation period of the parasite in the mosquitoes or increase the longevity of the mosquitoes, undesirable consequences may occur with an increased efficiency of these mosquitoes to transmit parasites to human. We therefore tested these hypotheses and experimentally produced different infection loads in vector mosquitoes Anopheles gambiae by using dilutions of Plasmodium falciparum isolates from naturally infected human donors. We observed that the longevity of the mosquitoes and the incubation period of the parasites were not affected by the parasite load. This is not consistent with the unintended risks that we investigated and thus strengthens the potential of transmission blocking interventions in the toolbox to combat malaria.

BackgroundEmerging artemisinin resistance and diagnostic resistance are a threat to malaria control in Africa. Plasmodium falciparum kelch13 (K13) propeller-domain mutations that confer artemisinin partial resistance have emerged in Africa. K13-561H was initially described at a frequency of 7.4% from Masaka in 2014-2015 but not present in nearby Rukara. By 2018, 19.6% of isolates in Masaka and 22% of isolates in Rukara contained the mutation. Longitudinal monitoring is essential to inform control efforts. In Rukara, we sought to assess recent K13-561H prevalence changes, as well as for other key mutations. Prevalence of hrp2/3 deletions was also assessed. MethodsWe genotyped samples collected in Rukara in 2021 for key artemisinin and partner drug resistance mutations using molecular inversion probe assays and for hrp2/3 deletions using qPCR. ResultsClinically validated K13 artemisinin partial resistance mutations continue to increase in prevalence with the overall level of artemisinin resistance mutant infections reaching 32% in Rwanda. The increase appears to be due to the rapid emergence of K13-675V (6.4%, 6/94 infections), previously not observed, rather than continued expansion of 561H (23.5% 20/85). Mutations to partner drugs and other antimalarials were variable, with high levels of multidrug resistance 1 (MDR1) N86 (95.5%) associated with lumefantrine resistance and dihydrofolate reductase (DHFR) 164L (24.7%) associated with antifolate resistance, but low levels of amodiaquine resistance polymorphisms with chloroquine resistance transporter (CRT) 76T: at 6.1% prevalence. No hrp2 or hrp3 gene deletions associated with diagnostic resistance were found. ConclusionsIncreasing prevalence of artemisinin partial resistance due to K13-561H and the rapid expansion of K13-675V is concerning for the longevity of artemisinin effectiveness in the region. False negative mRDT results do not appear to be an issue with no hrp2 or hpr3 deletions detected. Continued molecular surveillance in this region and surrounding areas is needed to follow artemisinin resistance and provide early detection of partner drug resistance, which would likely compromise control and increase malaria morbidity and mortality in East Africa.

BackgroundSystematic, long-term, and spatially representative monitoring of insecticide resistance in mosquito populations is urgently needed to quantify its impact on malaria transmission, and to combat failing interventions when resistance emerges. Resistance assays on wild-caught adult mosquitoes (known as adult-capture) offer an alternative to the current protocols, and can be done cheaply, in a shorter time frame, and in the absence of an insectary. However, quantitative assessments of the performance of these assays relative to the gold standard, which involves rearing larvae in an insectary, are lacking. Methodology/Principal findingsWe developed a discrete-time deterministic mosquito lifecycle model to simulate insecticide resistance assays from adult-captured mosquito collection in a heterogeneous environment compared to the gold standard larval capture methods, and to quantify possible biases in the results. We incorporated non-lethal effects of insecticide exposure that have been demonstrated in laboratory experiments, spatial structure, and the impact of multiple exposure to insecticides and natural ageing on mosquito death rates during the assay. Using output from this model, we compared the results of these assays to true resistance as measured by the presence of the resistance allele. In simulated samples of 100 test mosquitoes, reflecting WHO-recommended sample sizes, we found that compared to adult-captured assays (MSE = 0.0059), larval-captured assays were a better measure of true resistance (MSE = 0.0018). Using a correction model, we were able to improve the accuracy of the adult-captured assay results (MSE = 0.0038). Bias in the adult-capture assays was dependent on the level of insecticide resistance rather than coverage of bed nets or spatial structure. Conclusions/SignificanceUsing adult-captured mosquitoes for resistance assays has logistical advantages over the standard larval-capture collection, and may be a more accurate sample of the mosquito population. These results show that adult-captured assays can be improved using a simple mathematical approach and used to inform resistance monitoring programs. Author SummaryGrowing insecticide resistance in the mosquitoes that transmit malaria necessitates more widespread monitoring. Conducting assays on mosquitoes captured as adults is logistically simpler than raising them from eggs or larvae, the current recommended practice. However, this method is not widely used because survival when exposed to insecticide is known to depend on age and history of previous history as well as genetic resistance-factors that cannot be controlled when testing wild-caught adults. Here, we developed a mathematical model to quantify the difference in resistance measured via adult-capture assays compared to the gold standard larval-capture assays. We find that adult-capture assay results can be easily corrected using a formula based only on the measured resistance. This result has the potential to expand access to monitoring by reducing the time and infrastructure required to conduct these tests.

Plasmodium falciparum is the major cause of malaria in Africa, responsible for high morbidity and mortality across the continent. This study presents a systematic literature review and bibliometric analysis of P. falciparum research conducted in Africa between 2000 and 2024 as shown in Figure 1. Using the PRISMA framework, 10,903 peer-reviewed articles were retrieved from the Scopus database. Bibliometric analysis was performed using Bibliometrix in R and VOSviewer to assess publication trends, authorship networks, keyword evolution, and thematic concentration. Results reveal contributions from 18,345 authors across 4,903 journals, with an average of 31.83 citations per article. Research output has grown steadily over the two-decade period, with significant input from African scholars and international collaborators. The most active research themes include epidemiology, antimalarial drug resistance, vaccine development, vector biology, and socio-economic factors in malaria control. Despite this progress, the review highlights persistent gaps in genomic surveillance, localised insecticide resistance monitoring, and integration of social determinants into malaria intervention strategies. Regional disparities in research output remain, with some high-burden areas underrepresented. Collaboration among African institutions is limited compared to international partnerships. These observations indicate the urgent need for targeted funding, strengthened intra-African collaboration, and policies that contextualise malaria research within local health systems. Addressing these gaps is essential for speeding the continents malaria elimination agenda. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/681826v1_fig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1554b4aorg.highwire.dtl.DTLVardef@c21204org.highwire.dtl.DTLVardef@12cc70corg.highwire.dtl.DTLVardef@c016ab_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO Flow chart of Methodology using PRISMA method. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/681826v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@11056c9org.highwire.dtl.DTLVardef@1b1cfb3org.highwire.dtl.DTLVardef@baf4b7org.highwire.dtl.DTLVardef@18304b_HPS_FORMAT_FIGEXP M_FIG Graphical representation and summary of trends in Plasmodium falciparum in Africa. C_FIG

BackgroundRecent initiatives have promoted the application of genomic data to determine the trends and patterns of malaria transmission and the impact of interventions. This study aimed to evaluate and identify the most effective genetic metrics for monitoring the genetic diversity of Plasmodium falciparum and its correlation with malaria transmission intensities in Mainland Tanzania. MethodsA cross-sectional survey was conducted between February and July 2021 in 100 health facilities from 10 regions of Mainland Tanzania, categorized into four malaria transmission strata: high (two regions), moderate (two regions), low (three regions), and very low transmission strata (three regions). Dried blood spots (DBS) samples were collected from 12,875 symptomatic patients and all samples with positive results obtained by rapid diagnostic tests (n = 7,199) were sequenced using molecular inversion probes (MIPs). The MIPs targeted 1,832 single nucleotide polymorphisms (SNPs) distributed across the 14 P. falciparum chromosomes. Raw sequence data were analyzed using MIPTools and the filtered dataset was used to estimate different genetic metrics. ResultsAfter processing, 3,149 (43.0%) samples passed filtering conditions and were used for downstream analysis. The countrywide mean complexity of infection (COI) was 1.5, with 1,878 (59.6%) samples being monogenomic. The mean COI was significantly higher in high and moderate transmission strata (p < 0.001) compared to low and very low transmission strata. The odds of polyclonal infections were significantly lower in moderate (aOR = 0.67; 95% CI: 0.55-0.81; p < 0.001), low (aOR = 0.52; 95% CI: 0.43-0.63; p < 0.001), and in very low strata (aOR = 0.49; 95% CI: 0.40-0.61; p < 0.001) compared to the high transmission stratum. There was very little parasite genetic differentiation among regions with fixation index (FST) values ranging from 0 to 0.006. The countrywide mean pairwise identity by descent (IBD) was 0.02. The mean pairwise IBD by transmission intensity was similar with mean IBD = 0.0155 in very low, 0.0158 in low, 0.0156 in moderate and 0.0152 in high malaria transmission strata. Using discriminant analysis of the principal component (DAPC) parasite populations from different regions clustered within regions suggesting genetic similarity among them. ConclusionParasites from the sampled 10 regions had high complexity of infection and polyclonality, with a high correlation with regional malaria transmission intensities. Thus, these metrics can be potentially integrated with the current malaria surveillance and may be useful in the assessment of trends and patterns of malaria transmission. Further validation is needed to link these measures to current control strategies and evaluate how they might be used to determine the impact of different malaria transmission interventions in Mainland Tanzania.

Re-emerging of high malaria incidences in highlands of western Kenya pose a challenge to malaria eradication efforts. Anopheles coustani is a sub-Saharan mosquito species implicated in transmission of malaria in many parts of Africa as a secondary vector. It is a zoo-anthropophilic species that has been assumed to be of negligible importance. A cross sectional study was carried out in April to June, 2020 in Eluche location, Mumias East sub-County, Kakamega County, Kenya to establish the contribution of Anopheles coustani in malaria transmission. Pyrethrum spray collections (PSC) and Centers for Disease Control (CDC) and prevention light traps were used for sampling mosquitoes. Mosquitoes were collected from both indoors; between 0700h and 1100h using PSC and outdoors between 1800h and 0700h using CDC light traps. All mosquitoes were identified morphologically and female Anopheles heads and thorax were analyzed further using Polymerase Chain Reaction (PCR) for Plasmodium sporozoite. A total of 188 female Anopheles mosquitoes were collected from both PSC and CDC light traps. This constituted of; 80(42.55%) An. coustani, 52(27.66%) An. funestus, 47(25.00%) An. maculipulpis, 8(4.26%) An. arabiensis and 1(0.53%) An. gambiae. Malaria sporozoite detection was done to all the Anopheles female mosquitoes but only two An. coustani tested positive for Plasmodium falciparum. In conclusion, Anopheles coustani plays a major role in outdoor malaria transmission in Mumias East Sub-County of Kakamega County in Western Kenya.

Hyperlactatemia, a key marker of severe malaria, is closely linked to increased mortality, though the exact mechanisms remain unclear. It may result from increased lactate production due to tissue hypoxia or reduced lactate clearance from organ dysfunction. This study used Plasmodium yoelii 17XL (Py17XL) murine model of severe malaria, which closely mimics hyperlactatemia seen in human cases, to investigate the contributions of severe anemia and infection-related organ dysfunction to hyperlactatemia. Non-infectious anemia models were also included for comparison. Anemia was found to elevate lactate in both malaria-infected and non-infectious models, but Py17XL infected mice showed higher lactate levels, indicating that anemia alone doesnt fully explain hyperlactatemia. Evidence of tissue hypoxia, particularly in the liver, kidney, and gut, was seen with hypoxyprobe staining and upregulated hypoxia-inducible factor 1-alpha (HIF-1), suggesting that hypoxia drives increased glycolysis and lactate production. Impaired lactate clearance may also play a role, as infected mice showed signs of liver and kidney dysfunction accompanied by reduced clearance of 13C3-labeled sodium-L-Lactate. Whole blood transfusion combined with artesunate significantly improved lactate clearance compared to artesunate alone, underscoring the importance of addressing anemia in treatment. A link between intestinal damage and hyperlactatemia was suggested by correlations between trefoil factor 3 (TFF3), a marker of gut injury, and lactate levels in human samples. Our findings highlight the multifactorial origin of hyperlactatemia in malaria, driven primarily by anemia and tissue hypoxia, pointing to the need for therapies targeting both aspects to reduce mortality in severe cases.

Malaria, a tropical disease caused by Plasmodium and transmitted by Anopheles, remains a public health concern in Brazil. While most cases occur in the Amazon, transmission persists in the Atlantic Forest, where Anopheles mosquitoes of the Kerteszia subgenus are the primary vectors of human and simian malaria. Previous studies using cytogenetics, isoenzymes, and molecular markers have suggested cryptic species within Anopheles (Kerteszia) cruzii and Anopheles (Kerteszia) bellator. We sequenced 55 genomes: 35 An. cruzii s.l. (four with Nanopore and 31 with Illumina), 12 An. bellator s.l., and eight An. homunculus, the latter two with Illumina. Phylogenomic analysis revealed at least five cryptic species within An. cruzii s.l., labelled A-E, with evidence of sympatry in some locations. Anopheles bellator s.l. also forms a species complex, comprising at least three distinct lineages. These cryptic species showed high genetic differentiation (FST range: 0.4-0.7), typical of interspecific comparisons. In contrast, An. homunculus populations showed low differentiation (FST [~] 0.2), suggesting a single widespread species. Our analysis confirms cryptic speciation in An. cruzii and An. bellator, but not in An. homunculus. These findings are important for understanding malaria transmission in the Atlantic Forest, given that vector competence may differ among cryptic species.

Mass Drug Administration (MDA) is regarded as a potential strategy for locally interrupting transmission of human malaria under specific circumstances. However, insights on how MDA affects the eco-evolutionary dynamics of different Plasmodium species are not well known. We provide a computational model where the ecologically explicit life cycle of the parasite is implemented. Since the parasite inhabits two different ecological niches - human host and the mosquito - it undergoes different selection pressures during its reproduction. We use the model to perform an evolutionary analysis of the dynamics of resistance alleles under atovaquone, chloroquine and combined atovaquone-chloroquine drug treatments. Our study shows how the reduced viability of resistant parasites in the mosquito affects the spread of resistance and transmission interruption in treated human populations. Overall, results confirm that the disadvantage of drug-resistant genotypes in the mosquito vector is a good tool to achieve malaria control goals under MDA programmes.\n\nAuthor summaryEvery year there are millions of new malaria cases reported worldwide. The cause of the disease is the infection by Plasmodium, a protozoan which is transmitted between humans through the bite of a mosquito. Antimalarials have existed since long, but Plasmodium has evolved resistance to the treatment, making it necessary to develop new strategies to heal the infected humans. Lately, it has been pointed out that mosquitoes could be our allies when using drugs such as atovaquone, which resistant parasites have difficulties to reproduce in the mosquito. Here we study the scenarios in which these drugs, used in Mass Drug Administration (MDA) programmes, can interrupt the transmission of malaria in local treated populations.

The Bioko Island Malaria Elimination Project (BIMEP) has made significant progress in reducing the prevalence of Plasmodium falciparum on Bioko Island, Equatorial Guinea. However, like other malaria endemic islands like Sao Tome and Principe and Zanzibar, Tanzania, elimination efforts are hampered by imported infections. In an effort to understand the local transmission dynamics and the influence of importation on Bioko Islands P. falciparum population, whole-genome sequences were generated from field samples collected during the BIMEPs 2019 Malaria Indicator Survey (MIS). Within the sub-Saharan African context, we observed Bioko Island parasites did not significantly differentiate from nearby continental neighbors. Among Bioko infections, within-host diversity and the quantity of polyclonal infections appear similar to an area of moderate malaria transmission. However, we observed higher than expected genetic diversity among Bioko parasites, similar to high transmission areas, suggesting imported strains are contributing to transmission on the island. Among Biokos closest geographical neighbors, the flow of parasites with Bioko appeared more pronounced with the Gabonese parasite population, implying more importation may be coming from this region than others. Overall, despite significant investment in malaria control, results illustrate the challenges of eliminating malaria without both interrupting local transmission and accounting for importation from higher transmission areas, likely due to human migration. For there to be sustained progress towards elimination, the BIMEP needs, if feasible, to conduct targeted interventions of outgoing/incoming travelers, and ideally expand malaria control interventions to the continental region of Equatorial Guinea. ImportancePlasmodium falciparum accounts for the majority of malaria deaths globally, with over 500,000 estimated deaths in 2023, predominantly in sub-Saharan Africa, despite strong investment in control and elimination interventions. Incorporating sequencing technologies into malaria surveillance is viewed as a powerful tool to improve control strategies, including identifying parasite transmission pathways. Here, we provide the first genomic characterization of P. falciparum on Bioko Island and Equatorial Guinea since malaria control began in 2004, and use these data to better understand the contribution of neighboring regions to Biokos P. falciparum population. Results highlight the need to account for offisland contributors to transmission and to understand how unmitigated transmission in neighboring regions can hamper progress. This study furthers our understanding of how the flow of parasites between regions impacts infectious disease control and provides foundational genomic data in a previously undescribed region that can be used to inform malaria elimination efforts.

BackgroundPlasmodium vivax poses a major obstacle to malaria elimination because it can lie dormant in the liver for weeks or months before reactivating and causing a relapse of infection. These dormant forms (hypnozoites) cannot be detected using standard diagnostics, but recent P. vivax exposure and by proxy, hypnozoite carriage, can be inferred using antibody-based tests (serological markers). In this study, we examined how genetic variation in P. vivax affects the utility of these antibody markers, and whether redesigned antigens could improve performance. MethodsWe analysed global P. vivax genetic data to assess variation in leading serological markers. Based on this, we produced new antigen versions (haplotypes) that better reflect global sequence diversity, compared to the commonly used reference strain (Sal-1). Antibody responses against these new constructs were then tested using samples from well-characterised cohorts in Brazil and Thailand. Antibody levels were assessed in relation to how recently participants had a qPCR-detectable blood-stage P. vivax infection. We compared the ability of the haplotypes and reference constructs to correctly identify individuals infected within the prior 9-months. FindingsExtensive genetic diversity was identified in two P. vivax antigens, DBPII and MSP5. Several antigens had large numbers of circulating haplotypes globally, with the percentage with similar sequence identity to the reference Sal-1 ranging from 0.4% (MSP5) to 99% (S16). Two antigens exhibited strong differences in immunogenicity by region and construct (RBP2a and DBPII). However, for most proteins (5 out of 8), these differences had little impact on the accuracy of identifying recent exposure. In cases where performance was affected (e.g. RBP2a), this could be overcome by adding multiple antigens into the classification model. InterpretationEven highly diverse antigens can be effective serological exposure markers. Our findings highlight the importance of testing the impact of genetic diversity when designing serological tests and suggest practical strategies, such as using a mix of antigens, to ensure consistent performance across regions.

Drug resistance in Plasmodium falciparum threatens to undermine malaria control and elimination efforts. Senegal is a malaria-endemic country that has implemented successive antimalarial and chemopreventive drug-based strategies for two decades. Sulfadoxine-pyrimethamine (SP) is used for chemoprevention in Senegal for intermittent preventive treatment in pregnancy (since 2004) and SP plus amodiaquine (AQ) is used for seasonal malaria chemoprevention (SMC, since 2013). Using whole genome sequence (WGS) data from malaria patient samples from health facilities across Senegal (2006 - 2022), we observed near fixation of Pfdhfr triple mutant and fluctuation in Pfdhps and Pfcrt mutation frequencies over time. It is unclear how these mutations influence drug resistance and fitness phenotypes in natural isolates; therefore, we evaluated natural parasite isolates with different Pfcrt, Pfmdr1, Pfdhps, and Pfdhfr haplotypes. Parasites were culture-adapted and phenotyped for antimalarial drug susceptibility and competitive growth (fitness). Pfcrt CVIET + A220S + Q271E + N326S + R371I and Pfcrt CVIET + A220S + Q271E + I356T + R371I mutants were significantly more resistant to monodesethyl-amodiaquine (md-AQ) compared to Pfcrt wild-type (WT) and Pfcrt CVIET + A220S + Q271E + R371I mutants. Pfdhfr triple mutants were significantly more pyrimethamine (PYR) resistant than Pfdhfr WT and revealed a range of phenotypes, but this was not explained by Pfgch1 copy-number. Pfdhps A437G parasites were significantly more sulfadoxine (SDX) resistant compared to Pfdhps wild-type and Pfdhps S436A mutants, suggesting that A437G is a key mutation for SDX resistance. Competitive growth assays between Pfdhfr-Pfdhps mutants revealed that Pfdhps mutations do not always result in fitness costs. Ongoing phenotypic assessment and genetic validation of these mutations in a Senegalese background is necessary to assess the impact of drug pressure, identify evolving genetic determinants of drug resistance, and provide molecular markers for ongoing surveillance to monitor and guide the use of drug-based interventions. AUTHOR SUMMARYDrug resistance is a major concern for both preventing and treating malaria, especially in Africa where most malaria cases and deaths occur. Since 2013, Senegal has been giving children under 10 years old a combination of sulfadoxine-pyrimethamine plus amodiaquine to prevent malaria during the transmission season, called Seasonal Malaria Chemoprevention (SMC), and plans to continue expanding its use. However, there is evidence from genetic surveillance that drug resistance mutations are present in Senegal which could render this antimalarial drug combination ineffective. Here we use natural P. falciparum isolates obtained from Senegalese patients that represent the extant parasite population to evaluate the consequences of evolving mutations on antimalarial drug resistance and fitness phenotypes. This study is one of the first to use natural parasites to assess the impact of naturally derived mutations on drug resistance and fitness phenotypes. Our results provide evidence that certain combinations of drug resistance mutations impact both parasite drug resistance and fitness, and therefore need to be closely monitored and can inform optimal antimalarial combinations for the prevention or treatment of malaria. This work informs the ongoing evolution of resistance and fitness phenotypes in malaria endemic settings that are introducing new multi first line therapies (MFTs) and SMC interventions that have been used for decades in Senegal. Our approach creates a framework for using genetic surveillance data to form a hypothesis, which can then be phenotypically tested by measuring the resistance and fitness levels of genetically diverse natural parasite isolates.

Evidence based vector control interventions depends on understanding the distribution and the evolution of insecticide resistance in malaria vectors. This study aims to assess the status of insecticide resistances in Anopheles gambiae s.l., to pyrethroid insecticides such as permethrin, deltamethrin and alphacypermethrin from 18 study sites located in 15 districts across Rwanda, representing the key strata of malaria transmission in country. The larvae of anopheles were collected from October 2022 to April 2023 using dipping method, and reared to adult stage. The An. gambiae were exposed to alphacypermethrin 0.05% (Acyp_1X), permethrin 0.75% (Perm_1X), and deltamethrin 0.05% (Delth_1X) then tested using WHO bioassay standard protocol for insecticide resistance monitoring. As results, we found an extremely high resistance to permethrin in Gashora (Bugesera district), Mubuga (Karongi district), Rwaza (Musanze district), Kirarambogo (Gisagara district) with mortality rate of 42%, 48%, 69% and 70% repsectively. Subsquently, no recovery of susceptibility was observed in permethrin after pre-exposure to piperonyl butoxide (PBO) and observed mortality was 70% in Kirarambogo, while in Gashora, Mubuga and Rwanza it was 80% at each site. In city of Kigali, the mortality with alphacypermethrin was 94% and 93% with synergist PBO. Notably, in Kicukiro district we found that metabolic resistance mechanism was not driving the resistance mechanisms. These findings provide important understandings of pyrethroid resistance status in Rwanda and offering valuable insights for distribution of insecticide resistance intensity across country. This highlights the need for further studies to assess the spread of insecticide resistance and molecular driven resistance mechanism to address the issue in malaria vector control in Rwanda.

BACKGROUNDAngola ranks among the five countries with the highest malaria burden globally. The Ministry of Health in Angola has consistently partnered with international donors to sustain entomological surveillance and vector control strategies in a context of high malaria burden. METHODSVector surveillance was carried out in Luanda, Benguela, Namibe and Cuanza Sul provinces from 2016-2022. Collected adult mosquitoes were tested to assess the presence of Plasmodium parasites and determine blood sources. Larvae collections provided live material to test insecticide susceptibility in local Anopheles populations. Taxonomic determination of mosquitoes was based on external morphology and confirmed with molecular assays. The presence of Anopheles azevedoi was confirmed through morphology and genetic sequences, and errors in the original species determination were detected, discussed and corrected. OBJECTIVESThe study aimed to update the geographical range of Anopheles azevedoi in Angola and monitor the species susceptibility to public health insecticides. FINDINGS and MAIN CONCLUSIONSWe report on populations of Anopheles azevedoi occurring along the western coast of Angola, a highly abundant species with anthropophilic behavior in urban areas. Anopheles azevedoi is widely resistant to pyrethroids and DDT but fully susceptible to chlorfenapyr. We contribute with COI and ITS-2 barcoding sequences for future species identification and explain the reasons for which this species has been for long misidentified in Angola.

BackgroundNeonicotinoids are potential alternatives for targeting pyrethroid-resistant mosquitoes, but their efficacy against malaria vector populations of Sub-Saharan Africa has yet to be investigated. Here we tested and compared the efficacy of four neonicotinoids alone or in combination with a synergist against two major vectors of Plasmodium. ResultsUsing standard bioassays, we first assessed the lethal toxicity of three active ingredients against adults of two susceptible Anopheles strains and we determined discriminating doses for monitoring susceptibility in wild populations. We then tested the susceptibility of 5532 Anopheles mosquitoes collected from urban and rural areas of Yaounde, Cameroon, to discriminating doses of acetamiprid, imidacloprid, clothianidin and thiamethoxam. We found that in comparison with some public health insecticides, neonicotinoids have high lethal concentration, LC99, reflecting their low toxicity to Anopheles mosquitoes. In addition to this reduced toxicity, resistance to the four neonicotinoids tested was detected in An. gambiae populations collected from agricultural areas where larvae are intensively exposed to crop-protection neonicotinoids. However, adults of another major vector that occurred in urbanized settings, An. coluzzii, were fully susceptible to neonicotinoids except acetamiprid for which 80% mortality was obtained within 72 h of insecticide exposure. Importantly, the cytochrome inhibitor, piperonyl butoxide (PBO), was very effective in enhancing the activity of clothianidin and acetamiprid providing opportunities to create potent neonicotinoid formulations against Anopheles. ConclusionThese findings suggest that to successfully repurpose agricultural neonicotinoids for malaria vector control, it is essential to use formulations containing synergists such as PBO or surfactants to ensure optimal efficacy.