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.

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.

Background Malaria 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. Methods The 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 and above, residing in the study villages for 3 months and above, 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). Results Bayesian 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; 95percent credible interval [CrI]: 0.040 -- 0.112), while altitude had a protective effect (PM = -0.002; 9percent CrI: -0.003 to -0.001), with strong sub-village clustering of malaria infection (variance=0.485; 95percent CrI [0.333 -- 0.730]). In Buhigwe district (Kigoma region), shrub cover increased the risk of Plasmodium parasite prevalence (PM=0.119; 95percent 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; 95percent CrI: 0.117 -- 0.879) increased the risk of infection, with pronounced sub-village clustering (variance=0.84; 95percent CrI: [0.38 -- 2.40] ). In Nyasa district (Ruvuma), shrub cover had a modest positive effect (PM=0.070; 95percent CrI: 0.005 -- 0.135), in Muheza district (Tanga region), its effect was influential (PM=0.160; 95percent CrI: 0.056 -- 0.266). Risk maps revealed fine scale heterogeneity in the household level risk of Plasmodium parasite prevalence. Conclusion There 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.

Microsporidia sp. MB, a microsporidian symbiont found naturally in Anopheles mosquitoes, has potential as a novel malaria control tool since it can inhibit Plasmodium development and transmission. The most feasible MB-based Plasmodium control strategy would involve dissemination through live mosquito releases, or release of spores infective to mosquito larvae. To implement either strategy, establishment of stable mosquito colonies carrying MB at a high frequency is likely to be essential. The progeny of field caught An. gambiae s.l from Burkina Faso were isolated for individual egg laying and tested for MB. The progeny of the MB positive females were pooled and this process was repeated for multiple generations. The relative density of MB in different life stages and tissues of the An. coluzzii host was examined using a novel duplex qPCR assay. We also examined the impact of MB on fecundity through individualization for egg laying and counting of eggs. Finally, we examined laid eggs for presence of MB spores. Three An. coluzzii colonies and one An. gambiae s.l hybrid colony were established with high prevalence and density of MB and were maintained for more than two years with minimal intervention. MB prevalence and density was highest in eggs and adult females and lowest in L4 larvae; in adults density was highest in the gonads. Additionally, MB density increased in ovary samples following blood feeding which was likely due to the activation of sporogony. The production of spores is the reason why MB-carrying females lay more white non-hatching eggs and show a small reduction in fecundity. Establishment of several stable MB carrying An. gambiae s.l colonies and understanding the impact of spores on fecundity are significant steps forward in developing MB as a malaria control tool.

The spread of Anopheles stephensi into the Horn of Africa represents one of the main challenges for malaria control, given the species ecological plasticity and resistance to multiple insecticides. In response to the World Health Organizations 2022 vector alert, an adaptive, model-based spatial surveillance framework was developed and evaluated to improve detection, mapping accuracy, and operational responsiveness during invasion. Adaptive surveillance utilises initial observations to guide subsequent surveillance, linking the surveillance design to the underlying geographical characteristics of Anopheles stephensi distribution through observed data. This dynamic approach targets areas of high uncertainty and/or abundance, making the design responsive rather than predetermined. Focusing on Djibouti and selected regions of Ethiopia and Kenya, the adaptive surveillance was designed on previous in-country Anopheles stephensi surveillance data integrated with assembled open-source environmental, epidemiological, and demographic covariates. Key driver factors of the average monthly Anopheles stephensi catches varied geographically, although seasonality was universally important. Adaptive site allocation was optimised using a multicriteria target function which combines the trapping probability and uncertainty from previous surveys, with a simulation based on peaks-over-threshold (generalized Pareto) modelling of exceedances and Bayes factor-guided prioritisation. The selected adaptive surveillance design is the one that minimise the uncertainty in Anopheles stephensi trapping probability in hotspot areas. Optimal adaptive designs required between 50 to 59 sites per country, with uncertainty reductions in the probability of trapping projected up to 36% in Djibouti and more than 60% in Ethiopia and Kenya, with more than 60% site implementation halving uncertainty in Djibouti and Kenya and reducing it by up to 75% in Ethiopia. The proposed adaptive surveillance framework operationalises WHO guidance, accelerates hotspot identification, and inform targeted ecological studies and control interventions. It is extensible to other urban vectors (e.g., Aedes aegypti), enabling integrated, cross-border surveillance essential to contain Anopheles stephensi during ongoing invasion.

Successful transmission of malaria depends on the complex interactions between the Anopheles mosquito vector and the Plasmodium parasites. Plasmodium ovale, a neglected malaria parasite, successfully develops from ookinete to sporozoite within the Anopheles vector. To elucidate the molecular mechanisms underlying this interaction, we compared RNA-seq-based gene expression profiles of Anopheles gambiae infected with P. ovale and uninfected mosquitoes at 24 hours, 9 days, and 17 days post-infection. The results showed that 2,885 P. ovale transcripts were present only 24 hours after infection. During ookinete invasion (24 h post-infection), differential gene expression analyses revealed the up-regulation of genes related to metabolic processes and the down-regulation of genes associated with cytoskeletal activity in the mosquito. Notably, the non-immune genes with unspecific function AGAP003776, (Fold Change, FC 132.0), AGAP003777, (FC 88.3), and AGAP003778, (FC 104.1), Troponin C (Fold Change, FC 85) and Myofilin (FC 33.3) exhibited the most significant overexpression. Among the immune genes that were upregulated CTL3 (FC 55.9), CLIPB12 (FC 49.4), CTLMA5 (FC 14.5), TRYP7 (FC 24.4), CLIP C9 (FC 12.1) TRYP5 (FC 12.2), LRIM10 (FC 11.2), PPO6 (FC 7.7). This initial analysis of the interaction between P. ovale and An. gambiae identified several well-known candidates for transmission-blocking strategies, including LRIM1, APN1, and D7 family proteins. In addition, new potential candidates, including AGAP003776, AGAP003777, and AGAP003778 cluster, CLIPB12, LRIM10, the APN cluster, AGAP004860, ABCC9, CYP9K1 and GSTD3 were identified. These potential new candidate genes could play a significant role in the development of transmission-blocking strategies for An. gambiae infected with Plasmodium, particularly P. ovale. The urgent functional validation of these genes is required.

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.

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.

BackgroundElimination of Plasmodium vivax is challenging due to its dormant liver stages (hypnozoites), which can reactivate weeks or months after the primary infection, causing relapses and ongoing transmission of the parasite. Despite these challenges, P. vivax clinical case numbers have declined over the past decade in Cambodia. We used parasite genotyping to assess whether the decline in case numbers was reflected in parasite diversity and relatedness as a proxy to transmission. MethodsGenotyping was conducted on 182 symptomatic P. vivax isolates collected in eastern Cambodia in 2014, 2015, 2019 and 2023. A panel of 93 microhaplotype markers (vivaxGEN panel) was genotyped using Illumina sequencing. Population genetic measures were applied to determine infection diversity and relatedness (identity-by-descent (IBD)) each year. ResultsThe genetic results correlated well with clinical case numbers for the study years. The percentage of polyclonal infections was 5% in 2023 compared to 22-48% in earlier years (p<0.05) suggesting substantial reduction in superinfection and aligning with accelerated primaquine use in 2021. The cases in 2023 also had the highest percentage of infections with IBD >0.95 with one or more other infections (81.4% versus 8.9-10.8% in 2014-2019) indicative of inbreeding following population bottlenecking. In 2019, there was a spike in polyclonal infections (48%) and population diversity following local interruption of critical malaria control services. ConclusionsOur findings illustrate the potential of microhaplotype genotyping to inform on P. vivax transmission to assess intervention efficacy. In eastern Cambodia, the data provides evidence to support of widespread use of radical cure for patients with P. vivax malaria.

PurposeAnopheles stephensi is a malaria mosquito vector that has been raising international concern due to its invasive nature in Africa, including the nation of Djibouti. Since its initial detection in Djibouti in 2012, malaria morbidity and mortality have increased exponentially in the county. While there is an observed association increase in human malaria cases since the arrival of An. stephensi, high-quality evidence of An. stephensi carrying infective sporozoites is essential to determine the role of the invasive vector in malaria dynamics in Djibouti. This study seeks to confirm the link between An. stephensi and malaria transmission in Djibouti and examine genetic relatedness between Djiboutian An. stephensi populations and populations across the Horn of Africa. Such information regarding the An. stephensi populations and the Plasmodium species they transmit is necessary to devise appropriate control strategies and limit malaria transmission within and beyond the country. MethodsOne hundred and ninety-six adult An. stephensi mosquitoes from Djibouti were collected, molecularly confirmed, analyzed for a portion of the cytochrome c oxidase subunit 1 (COI), and tested for infective sporozoites using a highly sensitive and specific multiplex circumsporozoite enzyme linked immunosorbent assay (csELISA) bead assay. The COI sequences of one hundred and fourteen samples were further used to characterize the population genetic structure of the sampled An. stephensi and its genetic relatedness to other An. stephensi populations across the Horn of Africa. ResultsAll 196 samples were morphologically and molecularly confirmed to be An. stephensi. Plasmodium vivax210 sporozoites were detected with a positivity rate of 1.02%. An analysis of the COI region showed that the infected An. stephensi have the most prevalent COI haplotypes of invasive An. stephensi circulating in the Horn of Africa. ConclusionsThe findings from this study confirm the involvement of An. stephensi in P. vivax transmission in Djibouti and describe the genetic relatedness of Djiboutian An. stephensi populations to other populations across the Horn of Africa. This highlights the threat of An. stephensi invasion and supports a rapid and comprehensive response to mitigate the harm that An. stephensi populations cause, particularly through surveillance and control of adult populations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/707780v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@19f762corg.highwire.dtl.DTLVardef@7624forg.highwire.dtl.DTLVardef@c7492borg.highwire.dtl.DTLVardef@194c11f_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundThe widespread insecticide resistance increasingly threatens malaria elimination, prompting a reassessment of vector control strategies. As Tanzania transitions from standard pyrethroid-only insecticide-treated nets (ITNs) to new-generation nets, evaluating the impact of this shift on malaria transmission and resistance is critical. MethodsUsing the agent-based malaria model, EMOD, we assessed the impact of three ITN types, standard pyrethroid-only nets, pyrethroid-PBO nets (Olyset(R) Plus/PermaNet(R) 3.0), and the dual active, Interceptor(R) G2 nets (IG2) on malaria transmission and the evolution of insecticide resistance. We also evaluated different sequences for introducing the new-generation nets, and the impact of combining ITNs with indoor residual spraying (IRS). The model was calibrated using incidence and prevalence data from two regions in northwestern Tanzania, incorporating seasonality, insecticide resistance, and behaviors of dominant vectors Anopheles funestus (highly anthropophilic, endophilic) and Anopheles arabiensis (more opportunistic readily biting non-human hosts outdoors). ResultsChanging from standard pyrethroid-only ITNs to pyrethroid-PBO and thereafter to IG2 ITNs reduced homozygous-resistant An. funestus and An. arabiensis by 62.2% and 92.8%, respectively, and reduced incidence and prevalence by 94% and 75.2% respectively, under conditions where the probability of mosquito pyrethroid resistance was 0.75. Deploying IRS before the peak malaria transmission season in mid-May, in the second year following pyrethroid-PBO ITNs distribution, and repeating this every three years, reduced malaria incidence and prevalence by 76.4% and 52%, respectively. ConclusionIn contrast to continuous use of standard pyrethroid-only ITNs, which sustains resistance selection, transitioning to new-generation ITNs, with or without periodic IRS, can disrupt the evolutionary trajectory of pyrethroid resistance, reduce malaria burden, and strengthen progress toward elimination.

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.

BackgroundThe zoonotic parasite Plasmodium knowlesi is closely related to Plasmodium vivax, the second leading cause of human malaria. P. knowlesi line A1-H.1 can be maintained in human erythrocytes and is an experimental model for P. vivax biology. We present the first transcriptome time-course of the intraerythrocytic developmental cycle (IDC) of P. knowlesi A1-H.1 grown in human normocytes. ResultsBulk RNA-sequencing was performed at five IDC stages using tightly synchronised cultures, enabling identification of constitutively expressed genes and stage-specific biomarkers, and investigation of temporal expression patterns of multigene families. Comparative analysis with P. vivax orthologues revealed strong genome-wide conservation of temporal expression. Analysis of invasion-associated genes and ApiAP2 transcription factors identified genes with conserved expression patterns for which P. knowlesi is likely a good model to study the P. vivax gene. To support comparative analyses, we developed an open-access interactive webtool to explore and visualise P. knowlesi - P. vivax orthologue expression across the IDC. ConclusionsThis time-course dataset provides a reference transcriptome framework for P. knowlesi A1-H.1 and a resource for comparative Plasmodium biology. The interactive webtool facilitates rapid identification of candidate genes with conserved temporal expression, streamlining functional studies in which P. knowlesi A1-H.1 serves as a model for P. vivax.

Progress in malaria control has stagnated since the early 21st century in many countries, requiring new approaches such as the use of spatially-targeted interventions. Evidence on the effectiveness of spatially-targeted interventions is mixed. Their success can be dependent on whether the setting is endemic, the metrics used to target the intervention, and the spatial resolution and scale of deployment. We developed a two-age-class, spatially-explicit model of malaria at the community-scale for a district in southeastern Madagascar, accounting for environmental heterogeneity and human mobility. The model was fit to field-based case notifications and malaria prevalence data and then used to simulate three interventions: indoor residual spraying (IRS), long-lasting insecticide-treated nets (LLIN), and active case detection (ACD). We compared five spatial targeting scenarios for each simulated intervention: (i) equally distributed, (ii) targeting communities nearest or (iii) furthest from clinics, (iv) targeting communities with highest incidence, and (v) targeting communities that are spatially central. The non-targeted intervention was generally the most effective, but the least resource efficient. The second most effective intervention was based on spatial centrality, which reached a larger population while using fewer transportation resources than the equally distributed. No combination of interventions was able to eliminate malaria in the district, although a "perfect" ACD intervention could avert 100% of severe malaria cases. These results highlight the potential for targeted malaria interventions, especially in low-income settings, that take into account spatial structure in the human population and mobility to reduce malaria burden using fewer resources than conventional district-wide interventions.

BackgroundMalaria, transmitted by female Anopheles mosquitoes, remains a major public health challenge in Nigeria, where approximately 97% of the population is at risk. Despite large-scale investments, Nigeria continues to bear the worlds highest malaria burden. Long-lasting insecticidal nets (LLINs) are central to prevention, yet their effectiveness is increasingly undermined by non-usage, delayed replacement, and growing outdoor biting activity. National surveys (MIS, PMI) consistently report gaps in LLIN use, irregular implementation of the three-year replacement strategy, and persistent outdoor biting. This study quantifies the relative contributions of these behavioural and entomological factors to sustained malaria transmission across five Nigerian states. MethodsA deterministic compartmental model of malaria transmission was developed and calibrated using Bayesian inference with MCMC in CmdStanR. The model incorporated heterogeneous mosquito biting behaviour, LLIN effectiveness decay, and distribution cycles. Calibration used monthly malaria case data (2015-2024), demographic and entomological data (2015-2022), and DHS/MIS prevalence surveys (2015, 2018, 2021) for Akwa Ibom, Ebonyi, Kebbi, Oyo, and Plateau states. Counterfactual scenarios quantified malaria cases attributable to (i) outdoor mosquito biting, (ii) LLIN usage gaps, and (iii) delayed replacement. Parameter sweeps were used to assess how LLIN effectiveness changes with varying outdoor biting intensities. ResultsEliminating outdoor biting yielded the largest reductions in malaria incidence--Akwa Ibom (82.4%, 95% CrI: 74.0-89.6), Ebonyi (92.0%, 95% CrI: 87.5-95.5), Kebbi (76.4%, 95% CrI: 51.0-92.6), and Oyo (83.0%, 95% CrI: 74.9-89.6). LLINs sub-stantially reduced malaria transmission only under low outdoor biting intensities--below 1 bite per mosquito per month in Akwa Ibom and Ebonyi, below 0.2 in Kebbi and up to 0.8 in Oyo. In Plateau, outdoor biting contributed minimally (5.8%, 95% CrI: 4.8-6.3), while the gap between LLIN ownership and use was the dominant factor (23.1%, 95% CrI: 22.5-23.8), rising to 36.5% (95% CrI: 35.5-37.6) when combined with delayed replacement. ConclusionOutdoor mosquito biting is a dominant driver of persistent malaria transmission in Akwa Ibom, Ebonyi, Kebbi, and Oyo states, whereas low LLIN usage is the leading factor in Plateau. Although maintaining high LLIN coverage, adherence, and timely replacement remains critical, these efforts alone are insufficient where outdoor biting is widespread. Strengthening Nigerias malaria control strategy will require integrating LLIN deployment with targeted outdoor vector control and state-specific behavioural interventions to achieve sustained reductions in malaria burden.

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.

BackgroundMalaria transmission in Uganda is heterogenous, so the national malaria program needs information about the distribution of malaria to develop appropriate policies. While population-based community surveys estimate Plasmodium falciparum parasite rate (PfPR), they are too infrequent and sparse for routine malaria management. Health facility data is routinely collected and covers a large geographic scope, but the data is collected passively, variable in quality, and potentially highly biased. We aimed to triangulate test positivity rate (TPR) from health facility data to survey estimated PfPR data in Uganda to create monthly, high-resolution PfPR estimates. MethodsUsing matched health facility and survey data, we fit a multi-level logistic regression model that accounted for clustering at the district and region level, to predict PfPR from TPR. Additional covariates were explored to select a final model that reduced bias while prioritizing its utility for programmatic tasks. Model predictions were validated against observed PfPR and used to generate monthly district-level prevalence estimates from 2016 to 2024. Regional and national level estimates were made by weighting district level estimates by population. ResultsThe final model included a smoothed TPR term and proportion of severe malaria cases at a district-month level. Predicted PfPR was strongly positively correlated with the observed survey PfPR (Pearsons rank correlation rho =0.79, p<0.001). National estimates derived from predicted PfPR aligned well with survey estimates from the same time and area. ConclusionHealth Management Information System (HMIS) data, when paired with research data, can be used to estimate malaria prevalence with high spatial and temporal resolution. Estimates can be tested and models can be updated to help malaria programs best leverage facility data. In the context of declining survey frequency, HMIS-based modeling offers a resilient and cost-effective alternative for malaria surveillance and programmatic decision-making in Uganda and similar high-burden settings.

BackgroundLarval source management (LSM) was once central to malaria control before insecticide-treated nets and indoor residual spraying dominated. Renewed interest in LSM raises questions about its effectiveness in rural Africa, where habitats are dispersed, and vector species contribute unequally, and whether species-targeted larviciding could offer greater gains than broadcast approaches. MethodsThis modelling study quantified the potential impact of larviciding in African settings where multiple vector species contribute unequally to malaria transmission. We modeled malaria transmission in southeastern Tanzania using agent-based simulations incorporating seasonal dynamics, insecticide resistance, and semi-field biolarvicide efficacy. Outcomes were entomological inoculation rate, malaria incidence in under-fives, and operational larviciding costs. FindingsLarge-scale deployment of biolarvicides with >1-week residual activity substantially reduced malaria transmission, with disproportionately greater gains when control efforts were preferentially focused on the dominant vector species, Anopheles funestus, compared to broadcast approaches treating both An. funestus and An. arabiensis habitats. In the absence of ITNs, a four-month fortnightly larviciding campaign targeting An. funestus at 80% coverage reduced EIR by 58% and incidence by [~]40%, versus [~]55% incidence and [~]70% EIR reductions under broadcast strategies; targeting An. arabiensis alone yielded [&le;]30% EIR and [&le;]13% incidence reductions. Starting with pre-existing 80% ITN coverage, funestus-targeted larviciding further reduced peak EIR by [~]70% and incidence by [~]77%, versus [~]90% and [~]85%, respectively, with broadcast strategies, suggesting broadcast larviciding provided limited additional reductions beyond those achieved by the funestus-targeted approach. At 40% ITN coverage, additional reductions were [~]62% of EIR and [~]46% in incidence (funestus-targeted) versus [~]76% and 63%, respectively (broadcast). The targeted campaigns preserved a 30-50% cost advantage while sustaining >50% dry-season transmission reductions. Finally, high-coverage (e.g., 80%) funestus-targeted larviciding campaigns achieved greater impacts than lower-coverage (e.g., 40-60%) targeting both species. ConclusionsIn settings where multiple vector species contribute unequally to malaria transmission, preferentially targeting larviciding against the dominant vector species can deliver substantial epidemiological impact, with greater resource efficiency than broadcast approaches targeting multiple vectors. In Tanzania, where An. funestus drives most transmission; concentrating larviciding efforts on its characteristic aquatic habitats may offer a scalable, low-cost complement to established tools such as ITNs.

BackgroundPlasmodium vivax (P. vivax) has emerged as the primary cause of malaria in Cambodia. Achieving malaria elimination and securing malaria-free certification requires a focused effort on addressing P. vivax malaria. This is essential because the elimination of P. vivax often lags behind that of Plasmodium falciparum, making it a critical component in the overall strategy. This study assesses the feasibility of the Mass Drug Administration (MDA) and P. vivax Serological Testing and Treatment (PvSeroTAT) integrated with Reactive Case Detection (RACD) in two of the highest malaria burden operational districts of Cambodia and examines the potential for integrating these two approaches with existing malaria elimination efforts. MethodsThis study employs an observational, prospective cohort design. MDA with chloroquine (CQ) will be conducted in Stung Treng through four monthly rounds, while RACD with PvSeroTAT will be implemented in Sen Monorom, targeting households near confirmed P. vivax cases. Data on coverage, compliance, cost, and stakeholder perceptions will be collected through surveys, interviews, and malaria case monitoring. A Composite Feasibility Index will integrate quantitative and qualitative indicators. Cost and budget impact analyses will assess scalability for malaria-endemic districts. DiscussionInnovative and targeted public health approaches and tools are necessary to ensure the elimination of the malaria parasite reservoir, including the hidden hypnozoites. While MDA with CQ clears active blood-stage infections leading to immediate reductions in malaria prevalence, PvSeroTAT can detect past exposure to P. vivax by using serological markers allowing for targeted treatment of individuals at risk of developing relapsing infections with an 8-aminoquinoline. This helps reduce the parasite reservoir more efficiently. This study will provide insight into operational feasibility, implementation costs, community acceptance, and long-term sustainability. The findings will guide Cambodias malaria elimination efforts through improved surveillance and targeted interventions. Trial RegistrationOSF Preregistration: https://doi.org/10.17605/OSF.IO/5KZH7, retrospectively registered 15 October 2025.