Malaria Journal

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.

Malaria molecular surveillance (MMS) is becoming increasingly common in endemic settings and has been proposed as a tool for monitoring parasite transmission to inform programmatic decision-making. However, the conditions under which parasite genetic metrics provide interpretable signals for broader use cases, such as assessing intervention impacts and detecting importation, remain under-characterized. We present EMOD with Full Parasite Genetics (FPG), a simulation framework designed to explore how parasite genetic metrics arise from transmission, intervention, importation, and sampling processes at programmatically relevant timescales. Using seasonal scenarios across a range of transmission intensities, we demonstrate three principal findings. First, genetic metrics can detect insecticide-treated net intervention impacts at seasonal and yearly timescales, but the strength, timing, and form of the relationship between genetic and epidemiological measures vary by metric and sampling timing. Second, importation can break the expected relationship between parasite genetic diversity from local transmission intensity at very low incidence, allowing low-transmission settings with substantial importation to maintain elevated diversity metrics. Third, convenience sampling practices, including sample size, collection timing, and the clinical composition of sampled populations, introduce non-random biases in genetic metric estimation in a way that obscures the true transmission signal. Together, these findings show that parasite genetic metrics can support operational surveillance, but that their interpretation depends on transmission context, importation, metric choice, and sampling design. EMOD FPG provides a framework for evaluating these dependencies in future setting-specific analyses and for guiding the interpretation of parasite genetic data across sites and over time.

BackgroundMalaria remains a major public health challenge globally and in Tanzania, driven by persistent Plasmodium parasite transmission, environmental variability, and socio-economic inequalities. Despite targeted control strategies, transmission remains heterogeneous and under-captured by routine surveillance. This study utilised community cross-sectional surveys (CSS) data and spatial modelling to determine household-level risk estimates and identify micro-hotspots to guide more efficient, evidence-based malaria interventions in Mainland Tanzania. MethodsThe CSS data used in this study were collected in 13 villages across five regions with moderate to high malaria transmission in Mainland Tanzania between July and August 2023. Individuals aged [≥]6 months, residing in the study villages for [≥]3 months, were enrolled after providing informed consent and tested for malaria using rapid diagnostic tests (RDTs). Socio-demographic, clinical, anthropometric, parasitological and geo-coordinates data were collected using structured electronic tools. Household-level Plasmodium parasite prevalence was modelled using Bayesian geostatistical methods implemented through Integrated Nested Laplace Approximation within a Stochastic Partial Differential Equation framework, incorporating relevant environmental covariates. Model performance was evaluated using the Watanabe-Akaike Information Criterion (WAIC). ResultsBayesian models with village-specific covariates consistently outperformed null models, as indicated by lower WAIC values. In Kyerwa district (Kagera region), grass cover increased the risk of Plasmodium parasite prevalence (Posterior mean (PM)=0.076; 95% credible interval [CrI]: 0.040-0.112), while altitude had a protective effect (PM=-0.002; 95%CrI: -0.003 to -0.001), with strong sub-village clustering of malaria infection (variance=0.485; 95% CrI [0.333 - 0.730]). In Buhigwe district (Kigoma region), shrub cover increased the risk of Plasmodium parasite prevalence (PM=0.119; 95% CrI: 0.029-0.210) while in Ludewa (Njombe), both shrub (PM=0.512; 95% CrI: 0.066-0.989) and grass (PM=0.490; 95% CrI: 0.117-0.879) increased the risk of infection, with pronounced sub-village clustering (variance=0.84; 95% CrI: [0.38 - 2.40]). In Nyasa district (Ruvuma), shrub cover had a modest positive effect (PM=0.070; 95% CrI: 0.005-0.135), in Muheza district (Tanga region), its effect was influential (PM=0.160; 95% CrI: 0.056-0.266). Risk maps revealed fine-scale heterogeneity in the household-level risk of Plasmodium parasite prevalence. ConclusionThere was pronounced micro-scale heterogeneity in Plasmodium transmission across the study districts, driven by localised environmental factors and strong spatial dependence. Altitude had a protective effect, while vegetation cover increased the risk of infection. Geostatistical models effectively identified household-level hotspots, highlighting the limitations of aggregated surveillance, emphasising the need for locally precision-guided malaria control strategies to improve intervention efficiency and enhance the ongoing elimination strategies.

Introduction: Malaria during pregnancy is a major risk factor for low birth weight (LBW) in newborns, which in turn negatively affects the growth and development of the child. The World Health Organization (WHO) recommended interventions for pregnant women living in malaria endemic countries that include the use of intermittent preventive treatment in pregnancy (IPTp). However, WHO asserts that the coverage of pregnant women taking the recommended doses of IPTp are still very low. The primary goal of this study was to estimate the effects of increasing the coverage of doses of IPTp and to assess the effect of pregnancy timing in relation to seasonal transmission on malaria infections during pregnancy and neonates with LBW. We explored these effects in moderate and high transmission settings. Methods and Findings: A compartmental mathematical model depicting malaria during pregnancy with IPTp doses was formulated to analyze the effects of IPTp, insecticide treated net (ITN) use and seasonal variations in moderate and high malaria transmission settings. Our simulation findings suggest that increasing both ITN use and IPTp dose coverages to high levels, prevents 90% and 84% clinical cases for pregnancies starting in August in moderate and high transmission, respectively. Our model predicts that increasing the coverage of the first dose of IPTp to 90%, while lowering subsequent doses, averts 44% and 37% LBW cases for the August cohort in moderate and high transmission settings, respectively. Unprotected pregnancies overlapping the January peak in rainfall and malaria incidence during the third trimester experience the highest LBW burden. Conclusions: The highest IPTp coverage prevents the highest number of LBWs providing evidence of the benefits of scaling up IPTp. Overall, our results demonstrate that increasing ITN use has a substantial impact in reducing clinical malaria cases during pregnancy and improves birth outcomes. This highlights its importance as a key intervention, and the health benefits it would provide for malaria control goals for pregnant women. Pregnancies that overlap with the epidemic peaks in later trimesters lead to a rise in LBWs, indicating the necessity of protecting pregnant women at risk of malaria infection till delivery.

Gene expression analysis in malaria parasites has been used to define transcriptional regulatory networks but has been used less frequently to characterize parasite response to drug treatment or to show how parasites may evade killing. Here, we applied single-cell RNA sequencing (scRNA-seq) to hundreds of thousands of individually infected asynchronous red blood cells to evaluate the parasites response to treatment with three chemotypes that can be used for treatment (artemisinin) or prophylaxis and treatment (atovaquone, ganaplacide). We found that each treatment gave rise to different cell populations with different transcriptional profiles. Comparing single cell transcription patterns in compound-treated cells, to transcript patterns observed previously with synchronized cells showed an enrichment of cells expressing gametocyte-associated genes after artemisinin treatment but fewer lifecycle perturbations after treatment with the two other compounds. In contrast, bulk analysis showed an enrichment of pyrimidine biosynthesis transcripts for atovaquone treatment. Our results show that scRNA-seq may be used to profile diverse drug responses across many lifecycle stages and to potentially classify drug classes. ImportanceDetermining the mechanism of action (MOA) of compounds with antimalarial activity remains a key activity in both drug development and drug resistance studies but remains challenging for some chemotypes. Here we highlight the potential of single cell transcriptional sequencing to augment the process of MOA deconvolution. We develop a new analytical pipeline that involves comparing single cell transcription patterns to existing profiles from synchronized parasites to comprehensively characterize life cycle stage enrichments that may be observed after chemical perturbations. We also show that transcriptional feedback regulation may be present for some drug classes.

Malaria control programs are increasingly tailored at subnational scales; however, neighboring areas remain connected through human mobility, allowing parasite importation that may undermine independently timed interventions. Although the spatial targeting of control has been the focus of extensive research, the epidemiological consequences of temporal misalignment in intervention deployment across interconnected regions remain to be elucidated. We investigate how asynchronous timing of malaria interventions affects transmission dynamics using a two-patch susceptible-infected-susceptible metapopulation model. We compare synchronous and asynchronous intervention schedules and quantify their impact using measures of excess cumulative incidence attributable to asynchrony. The measure that will be used for this purpose is referred to as Asynchrony Induced Growth (AIG). Across a range of 10,000 parameter combinations, asynchronous implementation has been observed to result in a heightened incidence compared to synchronized deployment, though the impact is typically negligible in most endemic settings. Sensitivity analyses indicate that the impact is most significant when interventions are highly effective, infectious duration is brief, and transmission intensity approaches the elimination threshold. In such circumstances, asynchrony has the potential to substantially inflate case numbers, delay transmission interruption, or even prevent elimination entirely. In illustrative scenarios that reflect realistic settings, synchronizing interventions has been shown to avert large numbers of infections and shorten elimination timelines by years to decades. These findings demonstrate that, beyond spatial targeting, temporal coordination of interventions across connected areas can meaningfully enhance malaria control and elimination. Coordinated timing may be particularly valuable for cross-border or near-elimination programs and should be considered in operational planning and resource allocation.

Background Seasonal malaria chemoprevention (SMC) is a malaria intervention in which antimalarial drugs are administered monthly to children under 5 years of age during the high-transmission season. In the district of Moissala in southern Chad, SMC has been implemented since 2013, with an interruption in 2019, resumption in 2020, and expansion to five rounds of treatment in 2021. Recent World Health Organization (WHO) guidelines allow countries to adapt the timing and number of SMC rounds to local transmission patterns, creating a need to identify optimal strategies for each setting. In this study, we used mathematical modeling for three primary purposes: 1) to estimate the effectiveness of SMC in Moissala from 2018 to 2023, 2) to assess the impact of changes to SMC strategies since 2018, and 3) to determine the optimal SMC strategy in Moissala. Methods and findings We adapted a compartmental, climate-informed malaria transmission model to represent malaria dynamics in the presence of SMC. The model incorporates temperature and rainfall data to capture how climate variability influences malaria transmission over time. It was calibrated to routine surveillance data on malaria cases in children under five years old from 2018 to 2023. Using the calibrated model, we simulated malaria cases under alternative scenarios, including the absence of SMC and variations in the number and timing of SMC rounds. These simulations were then used to estimate the overall effectiveness of SMC, assess the impact of past changes in SMC strategies, and identify the optimal strategy in Moissala. Between 2018 and 2023, SMC reduced malaria cases in children under five by 26% (95% credible interval: 21%, 31%) relative to a scenario without SMC, corresponding to an average of approximately 14400 cases averted each year. The interruption of SMC in 2019 led to an estimated increase of 13600 cases (95% credible interval: 11200, 15800), representing a 31% rise during the high-transmission season. Expanding from four to five SMC rounds in 2021 reduced cases by 7% relative to a four-round schedule, while starting the five-round schedule earlier in June rather than July led to an additional 5% reduction. Overall, the most effective strategy from 2018 to 2023 was a five-round schedule beginning in mid-June. Conclusions Seasonal malaria chemoprevention has substantially reduced malaria incidence among children under five in Moissala. The currently implemented strategy of five rounds of SMC starting in June was estimated to achieve the greatest reduction in cases over the study period. Climate-informed modelling and open-source software can support timely decision-making across settings under changing climate and transmission conditions.

BackgroundMalaria poses a significant global health challenge, with over 200 million infections and around 500,000 deaths annually, predominantly affecting children under 5 in sub-Saharan Africa. Severe malarial anaemia (SMA), a major contributor to morbidity and mortality in this demographic, results from various factors including red blood cell destruction and immune-mediated clearance. The role of these mechanisms in SMA differs based on age and infection history. Recent studies indicate increased accumulation of P. falciparum in the bone marrow and spleen, highlighting the need for understanding localized host-parasite interactions. MethodsThis study examines the bone marrow response to malarial infection in young children with SMA or mild malarial malaria in Mozambique, contrasting it with responses observed in peripheral blood in the two cohorts. Results and conclusionsThe study demonstrated that SMA is associated with increased red blood cell production, iron metabolism and tissue injury, as well as higher total parasite biomass including peripheral and bone marrow parasitaemia. We also demonstrate that direct analysis of bone marrow aspirates provides far more resolution in stratifying host signatures and drivers of malarial anaemia across a spectrum of severity than systemic measures from blood samples.

The intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) remains the main strategy to prevent malaria in pregnancy. However, continued drug pressure may also contribute to the emergence of resistant parasites and impact the gametocyte carriage and subsequent infectiousness. Pregnant women are thought to be a potential reservoir for malaria transmission due to the increased carriage of gametocytes following long-lasting infections. We used molecular methods to examine 100 Plasmodium falciparum (P. falciparum) isolates collected from Mozambican women at delivery in 2014-15, to determine SP resistance polymorphisms in P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) genes as well as the presence of gametocytes by RT-qPCR. Overall, 54% and 7% of parasites harbored quintuple and sextuple pfdhfr/pfdhps mutant haplotypes, respectively. Gametocytes were detected in 34% of isolates. Gametocyte carriage was significantly associated with quintuple mutant infections (AOR = 7.5, p = 0.001), which accounted for 80% of infections with detectable gametocytes. Results indicate the relevance of ongoing surveillance of SP resistance in Mozambique to guide future evaluation of alternative IPTp approaches as resistance levels evolve and to anticipate potential implications for parasite transmission and maternal-fetal health.

Plasmodium falciparum merozoites invade erythrocytes using various ligand-receptor interactions. Important ligands encoded by the eba and Rh gene families have varying expression levels in different parasite isolates, affecting their vaccine candidacy. Analyses of clinical isolates from endemic areas in Africa have indicated that most variation in these expression profiles exists within each local area, and only minor differences are seen between areas, although comparisons with non-African populations have not previously been performed. To enable this, relative transcript levels of three eba genes and five Rh genes have been analysed in new population samples, Malaysian isolates sampled from Sabah State in Borneo prior to endemic malaria elimination, and Gambian isolates, cultured under the same conditions to harvest schizonts for reverse transcription quantitative PCR assays. Significant differences between these populations were seen for three of the ligand genes, levels of eba175 being higher in Malaysia, while levels of eba181 and Rh2b were lower in Malaysia. The gene transcript profiles did not differ between single genotype and or multiple-genotype isolates. The distinctness of the Malaysian population expression profile was also supported by comparing previous data on clinical isolates from Ghana. In tests for correlation with previously determined parasite multiplication rates, eba181 transcript levels correlated positively among Malaysian isolates but not among Gambian isolates. These findings suggest that expression of three P. falciparum merozoite ligands involved in invasion may be regionally differentiated, and further analysis of Asian parasite populations would be important if vaccines based on these candidates are to be considered for future use.

BackgroundThe malaria transmission potential and the vulnerability of Anopheles mosquitoes to different vector control methods depend, among other factors, on the endophily, endophagy, anthropophagy and survival of each species. Local information on these bionomic parameters is generally unavailable. MethodsTo address this, we estimated species-specific values of these parameters using an augmented version of the global database of bionomics data by Massey et al. (2016). We applied inclusion and exclusion criteria to select eligible studies with relevant experimental designs that minimise bias from collection methods for parous, sac, endophagy, and endophily rates as well as for the resting duration. For the human blood index (HBI), we separated data from indoor and outdoor collections. We fitted hierarchical Bayesian models with levels based on Anopheles taxonomy to estimate these quantities. Based on the estimated bionomics, we quantified the expected vectorial capacity reduction after the introduction of a pyrethroid-pyrrole insecticide-treated net (ITN) for 57 Anopheles species. ResultsWe identified 26 eligible studies for endophagy and 61 for the parous rate, leading to a Bayesian posterior average for the Anopheles genus of 42% (95% credible interval: 18-70) and 55% (32-77) respectively. HBI values widely varied depending on the location of collection, except for some species showing strong anthropophilic behaviours. Resting duration was estimated to be 2.1 days (1.2 - 4.8) at the genus level. Few studies were available to estimate the sac and endophily rates, which prevented us from deriving precise estimates for the whole Anopheles genus. Our estimates of the vectorial capacity reduction following the introduction of a pyrrole-pyrethroid ITN ranged between 48% and 76% across species, highlighting the important differences among mosquito species in vulnerability to vector control interventions. ConclusionThis work demonstrates how data from both Anopheles species complexes and individual species can be leveraged to generate species-specific estimates of bionomic parameters, capturing the local characteristics and behaviour of malaria vectors. The dataset is readily updatable as new data become available. However, more frequent and standardised field surveys are still needed to accurately characterise local vector behaviour.

BackgroundAnopheles gambiae sensu lato (s.l.) is the primary vector of malaria in sub-Saharan Africa. Although insecticide-based vector control has been central to prevention, the widespread emergence of insecticide resistance poses a serious biological threat to control efforts. Effective resistance monitoring is essential for sustaining vector control but remains highly limited in malaria-endemic hotspots. Here, we assessed pyrethroid and DDT resistance intensity and the frequency of the L1014F knockdown resistance (kdr) mutation in Anopheles gambiae s.l. populations from Sokoto, north-western Nigeria. MethodologyResistance status and intensity to five insecticides were determined in adult Anopheles reared from larvae collected in 2021 and 2022 using the World Health Organization (WHO) tube test and Centers for Disease Control and Prevention (CDC) bottle bioassay, respectively. A subset of resistant mosquitoes was analyzed using PCR-based diagnostic assays to identify species within the Anopheles gambiae complex and to genotype for the West African kdr mutation (L1014F). ResultsHigh knockdown times (KDT) were observed, with KDT50 ranging from 38 to 91 minutes and KDT95 from 104 to 678 minutes, indicating increased resistance levels across all insecticides. In 2021, resistance was detected to DDT, lambda-cyhalothrin, and permethrin, while susceptibility to alpha-cypermethrin (98%) and suspected resistance to deltamethrin (91%) were recorded. In 2022, a general increase in resistance to all insecticides was observed, with mortality rates ranging from 41% to 81%. High resistance intensity was observed against DDT, while permethrin and alpha-cypermethrin exhibited low resistance intensity in both years, failing to reach 10x the diagnostic dose. Deltamethrin and lambda-cyhalothrin showed low to moderate resistance intensity. The 1014F kdr mutant genotype was widely distributed (68.1%) across species and years. Allele frequencies were higher in An. gambiae s.s. (0.83) than in An. arabiensis (0.71), with significant deviations from Hardy-Weinberg equilibrium (p < 0.05), except for An. gambiae s.s. in 2021 (p = 0.7). ConclusionThese findings reveal a concerning increase in key insecticide resistance among Anopheles populations in Sokoto, underpinned by strong genetic mechanisms. This underscores the urgent need for integrated vector management strategies to sustain effective vector control efforts in the region.

BackgroundIn chronic asymptomatic Plasmodium vivax infections, the spleen accounts for more than 98% of total-body parasite biomass. Whether this splenic tropism also exists in acute infection and how the spleen influences pathogenesis have not been systematically explored. Materials and MethodsIn Papua, Indonesia, we compared plasma levels of P. vivax lactate dehydrogenase [PvLDH]) and circulating parasitemia in 24 spleen-intact and 25 previously splenectomized patients with acute uncomplicated vivax malaria. Clinical and hematology data were collected and plasma markers of intravascular hemolysis (cell-free hemoglobin [CFHb]), endothelial activation (angiopoietin-2), inflammation (interleukin [IL]-1 beta, IL-6, IL-18, IL-10, tumor necrosis factor-alpha) and neutrophil activation (elastase) were measured by ELISA. Giemsa-based histology in one spleen from an untreated patient splenectomized for trauma during an episode of acute vivax malaria enabled direct assessment of splenic and circulating parasitemia and biomass microscopically. ResultsCirculating parasitemia was 4-times higher in splenectomized compared to spleen-intact patients (median 21,100 vs 4,820 parasites/{micro}L, p=0.0002) but total-body P. vivax biomass (PvLDH) was 3-times lower in patients without a spleen (median 721 vs 2,140 ng/mL, p=0.026). Parasite staging and greater organ-specific symptoms suggest redistribution of parasites in the absence of a spleen. Linear regression modeling, adjusting for circulating parasitemia, patient age, sex and duration of fever, demonstrated an 8.1-fold higher PvLDH concentration in spleen-intact patients (95% confidence interval [CI]: 3.4-19.5-fold, p<0.0001), indicating a splenic biomass accounting for 89% (95%CI: 77.3-95.1%) of total-body parasites. Histopathology revealed a spleen-to-blood biomass ratio of 10.7, in-line with the PvLDH-based estimate. In spleen-intact patients, splenic P. vivax biomass correlated strongly with markers of disease intensity, endothelial activation and systemic inflammation, whereas circulating parasitemia correlated weakly or not at all. Compared to spleen-intact patients, CFHb, endothelial activation and systemic inflammation were higher in splenectomized patients while inflammasome-dependent responses were lower. ConclusionsP. vivax is predominantly an infection of the spleen, even in acute clinical vivax malaria. We conservatively estimate that 89% of total-body parasite biomass in acute infection is splenic. While the size of this hidden population correlates with disease intensity, the spleen likely regulates inflammatory pathways and heme-associated pathology.

Use of zooprophylaxis as a malaria control strategy has been recommended historically, but a complex relationship exists between animal ownership and malaria infection, with mixed associations described in the literature. We sought to characterize this relationship spatially and temporally in malaria-endemic regions of Africa. We used data from 392,843 individuals from 66 Demographic and Health surveys from countries within Africa to investigate the association between household animal ownership and Plasmodium infection. We used Bayesian models with Integrated Nested Laplace Approximation to incorporate spatially varying coefficient processes, allowing the association of interest to vary over space, time, and within strata of vector species occurrence, land cover, and number of animals owned by households. Spatially varying intercept models showed that ownership of cattle, chickens/poultry, goats, horses/donkeys/mules, pigs, and sheep was broadly associated with malaria infection, with odds ratios ranging from 1.55 to 1.67. However, spatially varying slope models revealed considerable heterogeneity, with odds ratio estimates for all animal types demonstrating both protective and harmful effects varying from 0.33 to 3.33 both subnationally and across time. We found no evidence that modification by vector species, number of animals owned, and land cover fully explained the variation in estimates. Unobserved localized cultural, behavioral, or ecological factors likely modify the association between animal ownership and malaria prevalence. Further exploring the nature of this relationship over space and time will be important to understanding how context-specific One Health dynamics between humans, animals and the environment affect malaria prevention and control efforts.

Background Vector control is essential to malaria control and elimination. National Malaria Programmes (NMPs) must make complicated decisions about vector control in the face of evolving epidemiology, biological threats like insecticide resistance, a growing vector control toolbox, and an increasingly constrained funding landscape. The WHO recently published a manual on subnational tailoring of malaria strategies, but limited efforts have been made to understand how NMPs prioritize data and factors that impact decision-making in practice. This study explores vector control decision-making processes, enablers, and barriers across 12 African malaria programmes. Methods We conducted semi-structured interviews with 13 NMP managers or designated representatives from 12 African countries. Interviews were conducted virtually via Zoom or in-person, audio-recorded, transcribed, and thematically analyzed using content analysis. Participants described the interventions in use, decision-making factors, stratification approaches, perspectives on new tools, and operational challenges. Results Insecticide-treated bed nets (ITNs) and indoor residual spraying (IRS) are the core interventions in all countries, with limited but growing use of larval source management, mainly larviciding. Vector control tool selection is driven by WHO guidance, resistance profiles and patterns, epidemiological trends, operational feasibility, and donor funding priorities. Sub-national stratification is widely applied; however, limited analytic and modeling capacity hinder consistent application. Gaps in entomological data result in incomplete data availability to guide stratification. New vector control tools were perceived as promising options, albeit constrained by cost, limited evidence, regulatory delays, and community acceptability. Funding emerged as the dominant driver of decisions, shaping intervention choices regardless of country preference. Participants emphasized substantial gaps in vector control protection related to residual transmission, outdoor biting, insecticide resistance, and unprotected populations living in temporary structures or associated with high-risk occupations. Conclusions Vector control decision-making among NMPs is shaped by an interplay of scientific evidence, operational realities, and external funding dynamics. Strengthening entomological surveillance, enhancing SNT analytic and model output interpretation capacity, securing sustainable financing, and improving community engagement are critical to advancing tailored deployment of tools. Decision-support frameworks that reflect the complexities facing NMPs may further enhance evidence-based, context-specific vector control planning.

Severe malaria disproportionately affects children during their earliest Plasmodium falciparum infections, when immunopathology rather than parasite burden often drives clinical deterioration. Because direct investigation of host-parasite interactions during severe disease is ethically impossible, we developed a two-dimensional ex vivo co-culture system that recapitulates key physiological features of malaria pathogenesis. Using PBMCs from malaria-naive and malaria-exposed adults co-cultured with a freshly adapted P. falciparum isolate, we modelled the combined effects of febrile temperature, pipecolic acid (PA), and lysophosphatidylcholine (LPC) depletion on IL-6 secretion. We also integrated clinical data from children with severe malaria in Anambra State, Nigeria. Across conditions, IL-6 output was not driven by temperature alone but by a metabolically gated interaction: febrile temperature amplified IL-6 only when PA was present, and LPC was not limiting. LPC depletion suppressed IL-6 to near-baseline levels regardless of temperature or PA, indicating that lipid availability constrains inflammatory signalling. Clinical data showed that adverse outcomes clustered with markers of multi-organ dysfunction. Together, these findings support a model in which IL-6 is a context-dependent mediator - participating in inflammatory pathways but not acting as a singular causal driver - and in which metabolic stress, febrile cues, and host tolerance mechanisms jointly shape cytokine production. Ongoing bioinformatics analysis will define the transcriptional responses of both parasite and host cells under these malaria-relevant conditions.

Background: Reliable inference of Plasmodium vivax recurrence states - relapse, recrudescence and reinfection (the ``3Rs'') - improves estimates of antimalarial efficacy. The R package Pv3Rs features a Bayesian model designed for P. vivax molecular correction, i.e., using parasite genetic data to infer recurrence states. The model is an extension of a prototype built to analyse microsatellite data from the Vivax History (VHX) and Best Primaquine Dose (BPD) trials. Methods: We re-analysed data from 212 VHX and BPD trial participants (493 recurrences) using Pv3Rs, comparing results with those from the prototype and with genetic relatedness estimated using Dcifer, a tool for estimating relatedness based on identity-by-descent. Posterior recurrence state probabilities were computed using both uniform and time-to-event priors: artificial but equal prior probabilities facilitate posterior interpretation, while time-to-event priors leverage all available information and enable re-computation of failure rates. Relatedness estimates were used to identify and correct instances of model misspecification. Results: The Pv3Rs model generated posterior probabilities for all recurrences and was able to jointly model data on all episodes per participant for 89% of participants, compared with 73% using the prototype. Recurrence state probabilities were broadly consistent across methods, though the Pv3Rs model elevated reinfection probabilities slightly. Relatedness estimates exposed various outliers consistent with half-sibling parasites and/or genotyping errors. Outlier correction impacted some per-participant failure probabilities, but reinfection-adjusted radical-cure failure rates of high-dose primaquine remained near 3%, in line with previous findings. Conclusion: Re-analysis of VHX and BPD P. vivax genetic data restates earlier reinfection-adjusted efficacy estimates. It demonstrates the increased computational capability and misspecification sensitivity of Pv3Rs, highlighting a need for careful analyses. Using relatedness-based diagnostics alongside model-based inference, we were able to harness the advantages of model-based inference and provide a framework for future P. vivax molecular correction.

BackgroundAnopheles stephensi, an invasive malaria vector originally endemic to South Asia, has rapidly expanded across East Africa. Its emerging malaria threats in urban Ethiopia threaten the elimination efforts, especially in areas once deemed low risk. A systematic review was conducted to synthesize the evidence regarding its bionomics and epidemiological impact, highlighting implications for urban malaria control strategies. This systematic review provides the first Ethiopia-specific quantitative evidence synthesis, addressing a vital knowledge gap necessary for guiding national malaria elimination programmes. MethodsWe conducted a PRISMA 2020-compliant systematic review and meta-analysis registered with PROSPERO (CRD420251176953). Searches of PubMed, Scopus, Web of Science, and regional repositories (2016-February 2026) identified studies reporting An. stephensi bionomics and epidemiological role in Ethiopia. Eligible studies required [&ge;]50% quality score on JBI appraisal tools. Random-effects meta-analysis estimated pooled proportions of An. stephensi among total Anopheles, with subgroup analyses by geography, habitat, and behavioural traits. Publication bias was assessed using Eggers and Beggs tests. ResultsEighteen studies (9 epidemiological, 11 bionomical) met inclusion criteria. The pooled proportion of An. stephensi was 0.51 (95% CI: 0.28-0.75) in epidemiological studies and 0.46 (95% CI: 0.26-0.66) in bionomics studies, with extreme heterogeneity (I{superscript 2} > 99%). Geographic analysis indicated significant variation: south-eastern Ethiopia showed a dominance of 0.73 (95% CI: 0.28-1.18) and eastern Ethiopia 0.57 (95% CI: 0.32-0.82), while central Ethiopia remained lower at 0.13 (95% CI: 0.12-0.14). These findings demonstrate genuine ecological differences rather than methodological objects, with substantial implications for region-specific vector management strategies. Extreme heterogeneity reflected genuine ecological variation across Ethiopia. No evidence of publication bias was detected. ConclusionAn. stephensi has very rapidly emerged as a major malaria vector in Ethiopia, with pooled proportions increasing from <10% at first detection (2016) to 51-73% in recent surveys (2024-2025), suggesting ongoing vector displacement parallel to invasion patterns documented in Djibouti. Geographic stratification indicates an urgent need for region-specific urban vector management integrating larval source management, resistance monitoring, and community engagement, particularly in south-eastern Ethiopia, where near-complete vector replacement has occurred. Author SummaryMalaria is usually thought of as a rural disease, but a new mosquito species called An. stephensi is changing that picture in Ethiopia. Originally found in South Asia, this mosquito has spread quickly across East Africa and is now common in Ethiopian towns and cities. We reviewed and combined results from published studies to understand how this species behaves and how much it contributes to malaria transmission. Our analysis shows that An. stephensi has become one of the dominant malaria vectors in several regions of Ethiopia, especially in the east and south-east, where it has almost replaced other mosquito species. This rapid change means that malaria risk is increasing in urban areas that were previously considered low risk. These findings highlight the urgent need for new control strategies that focus on city environments, such as managing breeding sites, monitoring insecticide resistance, and involving communities in prevention efforts. By understanding how An. stephensi is spreading and adapting, we can better protect urban populations and support Ethiopias malaria elimination goals.

BackgroundThe World Health Organization (WHO) has raised concerns over increasing Pfhrp2/3 deletions, undermining the sensitivity of Pfhrp2-based rapid diagnostic tests (RDTs). Close monitoring of the population and a change in diagnostic methods are recommended if the prevalence of parasites with Pfhrp2/3 deletions exceeds 5%. In high transmission settings, accurate estimates are hampered by the frequent occurrence of mixed-clone infections (multiplicity of infection; MOI). Objective and MethodsIf parasites with and without deletions are present in an infection, standard molecular assays cannot detect the presence of the former. To accurately estimate frequencies of haplotypes with Pfhrp2/3 deletions in the presence of mixed infections, a novel statistical model that combines genetic/molecular information from Pfhrp2/3 with that from neutral markers is introduced. Maximum-likelihood estimates (MLEs) are obtained for haplotype frequencies characterized by markers at Phrp2/3 loci and loci for neutral markers. The expectation-maximization algorithm is used to derive the MLEs. The adequacy of the method (precision and accuracy) is assessed by numerical simulations. ResultsThe method was applied to an active surveillance study conducted in a tribal community in Jagdalpur, India, which enrolled febrile community members (n = 432) between October and November 2021. Four markers each at Pfhrp2 and Pfhrp3 are combined with one marker each at Pfmsp1 (which encodes P. falciparum merozoite surface protein 1) and Pfmsp2. Data from a total of 117 patients who had both P. falciparum infections and genetic information for the molecular markers underwent further analysis with the novel statistical method. ConclusionResults indicate that this novel method has promising statistical properties (asymptotic and in finite samples) and can be readily applied to real-world situations. A stable implementation of the method in R is provided. This novel approach enables accurate estimation of Pfhrp2/3 deletion frequencies in complex P. falciparum infections, addressing a key limitation of current molecular surveillance methods. Author summaryPlasmodium falciparum (Pf) causes the most severe form of human malaria, accounting for over 90% of cases. Rapid diagnostic tests (RDTs) have become a cornerstone of malaria control. These RDTs detect Pf-specific antigens in a blood drop. HRP2/3 emerged as the best antigen for such tests because it is Pf-specific and expressed in abundance. However, some parasites lack the genes that code for HRP2/3 proteins. If parasites in an infection have such gene deletions, RDT results can be false negative. The WHO considers the containment of such deletions a public health priority and recommends monitoring their prevalence. The detection of HRP deletions is challenging if parasites with and without deletions co-occur in infections because standard molecular assays cannot detect deletions in this situation. To overcome this challenge, we introduce a novel statistical method to estimate the frequency distribution of parasite variants with deletions. The method combines information from neutral molecular markers and from HRP-related markers to correct for unobservable information. Here we provide a derivation of the statistical model, a stable implementation, and test its statistical properties with synthetic and real data, thereby showing that our method is well-suited for the underlying problem.

Rectal artesunate (RAS) is a pre-referral intervention recommended for children with suspected severe malaria in remote settings where injectable treatment is not readily available. Although clinical trials have demonstrated efficacy, less is known about the behavioural and health system factors influencing effectiveness under routine conditions. A convergent parallel mixed-methods design was used to assess implementation of Zambia's RAS intervention package across three districts: Serenje, Chama, and Mwinilunga. A retrospective case-tracking investigation of all 300 children with suspected severe malaria recorded by community health workers (CHWs) assigned to study facilities examined progression and attrition across the severe malaria care cascade. In-depth interviews and focus group discussions with caregivers, CHWs, and other stakeholders explored barriers and facilitators influencing progression. Among 300 enrolled children, early attrition occurred due to negative rapid diagnostic test results. Of 239 RDT-positive children, 218 (91.2%) received RAS. Referral completion was lower; among 261 children referred and followed up at health facilities, 209 (80.1%) were confirmed to have completed referral. Of 186 children diagnosed with severe malaria at the facility, 167 (89.8%) received both injectable artesunate and follow-on artemether-lumefantrine. Patterns of disengagement varied by district, with Serenje demonstrating the most consistent progression, Chama experiencing the largest drop-off at RAS administration, and Mwinilunga showing the lowest completion of follow-on treatment. Qualitative findings revealed strong community appreciation for RAS despite stockouts, alongside social and behavioural barriers, including gendered responsibilities, transport challenges, and confusion following symptom improvement, that discouraged referral completion. RAS can be a life-saving intervention when embedded within strong health systems and community structures. Zambia's experience underscores the need for comprehensive implementation strategies that extend beyond drug distribution to include sustained CHW training, reliable commodity supply, functional referral systems, and meaningful caregiver engagement.