Malaria Journal

Background: Malaria remains a major cause of morbidity and mortality among children under five years in Ghana. This study examined household, maternal, and child characteristics associated with malaria prevalence. Methods: Data were drawn from the 2019 Ghana Malaria Indicator Survey. The analytic sample comprised 2,867 children with valid malaria rapid diagnostic test results. Analyses accounted for the survey's multistage cluster design, including primary sampling units, strata, and sampling weights. Survey-weighted descriptive statistics, Rao-Scott corrected chi-square tests, and survey-weighted logistic regression with a quasibinomial link were used. Results: Overall, 22.9% of children tested positive for malaria. At the bivariate level, child age, number of children under five, household wealth, residence, region, household net usage, electricity, television ownership, maternal education, household size, and anaemia level were significantly associated with malaria status (p < 0.05). In the fully adjusted model, children in the poorest and poorer households had higher odds of testing positive than those in the richest households (AOR = 4.13 and 3.13, respectively). Rural children had higher odds than urban children (AOR = 2.36). The highest regional odds were observed in the Eastern Region (AOR = 15.3) compared with Greater Accra. Older children and those with severe anaemia had higher odds of testing positive (AOR = 4.17 and 24.5, respectively). Maternal education significantly interacted with household wealth and household net usage. Conclusion: Household wealth, region, residence, maternal education, child age, and anaemia level were important correlates of malaria prevalence. Findings support interventions addressing socioeconomic and regional inequalities in childhood malaria.

Background: Community case management has been scaled up nationally in Zambia over the last decade. However, there is limited evidence on how this nationwide implementation has affected febrile patients' access to malaria diagnosis and treatment in Zambia. Methodology: This study analyzed four rounds of Malaria Indicator Survey (MIS) data (2012-2021) to evaluate: 1) proportion of all-ages individuals with fever who sought treatment from a formal provider, 2) proportion of individuals going to CHWs over time, among those who sought treatment at a formal provider, 3) time duration between fever onset and treatment seeking at a formal provider, and 4) proportion of children <5 with malaria that received Artemether-Lumefantrine (AL) treatment. Mixed-effect logit models were employed to examine determinants of treatment-seeking behavior and factors affecting AL receipt among children <5 with malaria cases. Results: The proportion of febrile patients seeking treatment remained below 60% throughout 2012-2021, and AL receipt among children with malaria cases consistently stayed below 50%. The mean interval between fever onset and initial treatment-seeking encounter decreased from 2.42 days in 2012 to 1.71 days in 2021. Among formal care seekers, CHW utilization increased from 1.5% in 2012 to 10.0% in 2018 before declining to 3.2% in 2021. Longer walking time to the nearest health facility was associated with lower odds of treatment seeking, whereas CHW density was not associated with treatment seeking or AL receipt. Children who did not went to formal providers had lower odds of AL receipt than those who sought treatment from CHWs. Conclusion: Despite nationwide CCM scale-up over the last decade, significant barriers persist in malaria patients' access to diagnosis and treatment in Zambia. Our results indicate that while CCM coverage should be maintained and further expanded, additional complementary interventions are also needed to overcome remaining access barriers.

Insecticide-treated net (ITN) use - defined as the proportion of a population that use ITNs - is a measure of ITN uptake that is used in the estimation of malaria burden and evaluation of intervention programs. However, binary classification of individuals as users or non-users does not account for variations in ITN-related protection attributed to deleterious factors such as chemical and physical degradation, and increased insecticide resistance in vector populations. In this paper, we present a parsimonious model for malaria dynamics in mosquito-human populations in the presence of varying ITN use conditions. Using this model, we propose a new standardised measure of ITN coverage termed the "efficacy-adjusted use" defined as the equivalent level of use, assuming fully efficacious nets, that would be required to achieve the same level of theoretical EIR reduction. This more nuanced measure is used as a proxy for studying ITN-attributed protection across 44 African countries. We find that estimated protection levels in current ITN paradigms is significantly lower than indicated by crude ITN use metrics, with insecticide resistance having the largest deleterious effect. Furthermore, recent adoption of next-generation nets is estimated to have mitigated a 13% reduction in protection compared to a counterfactual pyrethroid only scenario.

Prolonged use of the few insecticide classes available for long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) has driven widespread physiological resistance of malaria vector mosquitoes to this limited arsenal of active ingredients. However, recent innovations like next-generation LLINs (NG-LLINs) containing two complementary insecticides and new insecticide classes for IRS offer new opportunities for pre-emptive resistance management by deploying more diversified actives as mixtures, combinations, rotations or mosaics. Here a deterministic model of mosquito foraging behaviour was formulated to predict the probabilities of deterrence, mortality or successful feeding across repeated feeding attempts in scenarios with different combinations of NG-LLINs and/or IRS micro-mosaics with varying levels of insecticide diversification between neighbouring houses. Final fates were classified based on whether or not the mosquito eventually died or successfully fed, and whether the latter occurred indoors or outdoors after exposure to zero, one or several IRS insecticides. The primary outcome was the probability that a single F mosquito carrying a novel resistance trait to a new IRS insecticide successfully feeds, survives and reproduces, thereby establishing those traits within the population. The secondary outcome was the selection coefficient governing the spread of such novel resistance traits from the F generation onwards. For highly anthropophagic and endophagic vectors like Anopheles funestus, combining NG-LLINs with IRS micro-mosaics using two insecticides may reduce emergence rates for novel resistance traits against IRS insecticides by approximately 2 to 2.5-fold, mainly through direct killing by NG-LLINs, although exposure to both IRS actives when forced to visit multiple houses also contributes to a lesser extent. However, such resistance management benefits are fundamentally constrained by outdoor feeding behaviours that limit or completely prevent indoor insecticide exposure. Increasing IRS micro-mosaic insecticide diversity beyond two actives is unlikely to further dampen resistance emergence rates because few mosquitoes survive long enough without feeding to encounter several IRS treatments. Once a resistance trait becomes established in the vector population, selection coefficients remain consistently high enough to force the spread of those traits, regardless of intervention combination. For more exophagic, zoophagic vectors like An. arabiensis, NG-LLINs plus IRS micro-mosaics are not expected to provide any meaningful resistance management benefit because frequent outdoor feeding, often on animals, allows them to largely avoid insecticide exposure altogether. Exclusively indoor-focused vector control strategies may not satisfactorily slow insecticide resistance emergence and spread, so new outdoor protection measures that close these coverage gaps with complementary insecticides will be needed.

Malaria remains a significant global health challenge. Intermittent Preventive Treatment of malaria in school-age children (IPTsc) is recommended to reduce disease burden, but optimal drug choice is unclear. Dihydroartemisinin-Piperaquine (DP) is highly efficacious but there are concerns about widespread use given emerging artemisinin resistance and its role as an alternative first line treatment. Thus, non-artemisinin alternatives to DP are needed. 646 Malawian primary school children participated in the second stage of a 3-arm randomized controlled trial. Participants were allocated to IPTsc with 1) DP, 2) Sulfadoxine-Pyrimethamine+Chloroquine (SP+CQ) or 3) Control (no treatment). Study drugs were administered at three six-weekly visits. Outcomes were measured 6-8 weeks later. The primary outcome was Plasmodium falciparum (Pf) prevalence detected by qPCR. Secondary outcomes included clinical malaria and anemia. Analysis was modified intention-to-treat. Outcome assessment included 588 (91%) participants. Prevalence of Pf infection was 18% (34/198) in the IPTsc-DP arm, 27% (52/200) in the IPTsc-SP+CQ arm, and 48% (89/190) in the control arm. Compared to control, both IPTsc-DP (adjusted Odds Ratio [aOR] 0.22, 95%CI:0.14-0.36, p<0.001) and IPT-SP+CQ (aOR 0.38, 95%CI:0.24-0.59, p<0.001) significantly reduced odds of infection. Both regimens also decreased anemia (DP: aOR 0.45, 95%CI:0.21-0.93, p=0.035; SP+CQ: aOR 0.47, 95%CI:0.22-0.98, p=0.048) and clinical malaria (DP: adjusted Incidence Rate Ratio [aIRR] 0.41, 95%CI:0.28-0.60), p<0.001; SP+CQ: aIRR 0.60, 95%CI:0.43 - 0.84, p=0.003). In Malawi and settings with similar malaria drug resistance profiles, SP+CQ may be a suitable alternative to DP for IPTsc. Clinical Trial Registration ClinicalTrials.gov ID: NCT05980156

The movement of people across country borders has implications for malaria control and elimination. Namibia is a low transmission country in southern Africa that borders two high transmission countries, Angola and Zambia, yet the extent to which cross-border connectivity constrains progress toward elimination remains unclear. In this study, we aimed to explore the feasibility of pre-elimination in Namibia, accounting for local transmission dynamics, climatic variability, international connectivity and current intervention coverage levels. A compartmental mathematical metapopulation model of malaria transmission was used to estimate the change in cases relative to the present status quo. Our findings suggest that Namibia could achieve pre-elimination status by 2034 through robust cross-border management targeting 50% of migrants and travelers while simultaneously scaling up the effectiveness of vector control interventions across Angola, Namibia, and Zambia. Within a coordinated multi-country approach, managing cross-border travel without additional interventions reduces Namibias case burden by up to 33% over 10 years. In contrast, isolated national strategies were insufficient to offset importation pressure from neighbouring high-transmission settings. Cross-border management poses challenges but is necessary for elimination in low-transmission settings. Simulated insecticide resistance resulted in marginal increases in incidence rate in Angola and Zambia, indicating possible health system resilience to increasing insecticide resistance. Overall, this study provides a quantitative framework for regional malaria policy, shifting from isolated national efforts to a synchronised, multi-country approach to achieve elimination in low-transmission settings.

Transmission-blocking vaccines (TBVs) are a promising strategy to reduce malaria transmission by targeting parasite stages within the mosquito. However, parasite genetic diversity may limit vaccine efficacy. We used next-generation amplicon deep sequencing to identify non-synonymous single nucleotide polymorphisms (SNPs) in Pfs25 from 184 Plasmodium falciparum isolates from Senegal, Tanzania, Ghana, and Burkina Faso. Prioritized SNPs were introduced into P. falciparum via CRISPR-Cas9. For the G116C variant, gametocyte development was evaluated by microscopy and qPCR, and mosquito infectivity was assessed by SMFAs. We identified 26 SNPs, including 24 novel variants. Functional assays showed that the Pfs25 G116C mutation did not affect gametocyte development or exflagellation. SMFA showed no significant differences in oocyst prevalence or intensity between mutant and WT parasites. These findings highlight the importance of integrating genetic surveillance with functional validation to guide the development of effective transmission blocking interventions

Malaria elimination is shaped by complex interactions among climatic, environmental, socioeconomic, demographic, health-system, and intervention-related factors. However most studies examine only subsets of these drivers, limiting understanding of their combined influence on epidemiological risks. In this study, we integrated 25 years of data from 44 African countries on malaria burden and control, climate, environmental and land-use conditions, socioeconomic and demographic characteristics, and health-system capacity within a unified geospatial, explainable machine-learning, and forecasting framework to characterize spatiotemporal patterns of malaria, quantify the relative contributions of key determinants, and generate 10-year Africa-wide and country-specific forecasts of malaria incidence and mortality rates per 1,000 people at risk. We identified and mapped malaria incidence and mortality hotspots using the Getis-Ord Gi* statistic. Our analyses showed that both incidence and mortality burden remained highly heterogeneous across Africa, with persistent hotspots concentrated in the West and Central Africa. The explainable machine-learning model, that achieved high predictive performance (i.e., XGBoost for incidence, holdout R$^2$ = 0.92; Random Forest for mortality, holdout R$^2$ = 0.91), identified lower availability of hospital beds (per 1,000 people), higher mortality rate attributed to unsafe WASH (per 100,000), and lower percentage (\%) of people using handwashing facilities as the top three most influential determinants of higher risk of infection across Africa whereas higher mortality rate attributed to unsafe WASH (per 100,000), lower \% of people using at least basic sanitation services, and access to electricity (\%) were associated with worse mortality outcomes. Forecasting models also demonstrated strong predictive accuracy (Naive persistence and Elastic Net, holdout R$^2$ = 0.98 for incidence and 0.97 for mortality). Assuming current intervention and structural conditions persist, Africa-wide malaria incidence was projected to remain broadly stable, with a modest upward trend by 2035, whereas mortality was projected to decline initially and subsequently remain relatively unchanged. However, substantial country-level heterogeneity showed both emerging transmission hotspots and persistently high-burden countries. this suggests there is a need for sustained control and accelerated elimination efforts. Overall, this study demonstrates that integrating geospatial analysis, explainable machine learning, and forecasting provides a robust framework for understanding malaria dynamics, identifying the key determinants of burden, anticipating future trends, and supporting geographically targeted malaria control across Africa. Beyond malaria, our analyses can be applied as a generalizable approach for infectious disease surveillance, early-warning systems, hotspot detection, resource prioritization, and precision public health using large-scale longitudinal health data.

Malaria programmes increasingly rely on modelled evidence to support intervention prioritisation, resource allocation, and elimination planning, yet a persistent gap remains between technical modelling outputs and their use in decision-making. We describe the design, implementation, and early outcomes of the Regional Malaria Modelling Translational Fellowship, a six-month executive education programme delivered in 2025 to 21 fellows nominated by national malaria programmes in seven African countries. The Fellowship was designed to strengthen translational capacity by focusing on question formulation, systems thinking, model design and critique, interpretation of outputs, uncertainty, health economics, communication, and stakeholder engagement. The hybrid structure combined three intensive in-person blocks with regular virtual sessions. Country-teams work on capstone projects throughout the Fellowship, applying learnings to develop policy-relevant modelling proposals aligned with national malaria priorities. The programme was accredited as a University of Cape Town short course, which supported credibility, participant commitment, and institutional endorsement. Early evaluation showed improvements across all competency domains, with the largest gains in fellows' confidence in applying modelling to decision-making, translating model findings into recommendations, and communicating technical results to non-technical audiences. Qualitative feedback suggested that the Fellowship helped shift participants' engagement with modelling from passive acceptance of results toward critical interpretation, collaborative dialogue, and practical application. These findings suggest that translational, executive-style training can strengthen the interface between modelling and malaria policy. The publicly available curriculum offers a replicable framework that may be adapted for other infectious disease and public health settings where modelling evidence is increasingly central to decision-making.

A common barrier to insecticide-treated net (ITN) use, the backbone of malaria prevention, is heat discomfort. Using multilevel regression models, we quantified the association between temperature and ITN use reported in Demographic and Health and Malaria Indicator Surveys from 2011-2022 across 28 countries in sub-Saharan Africa. Marginal predicted probability of use was unimodal with nighttime temperature and decreased above a threshold daytime temperature. For women (n=731,947), temperatures above specified thresholds were associated with lower use, with adjusted odds ratios [aOR] 0.91 for daytime (95% confidence interval [CI] 0.88-0.94) and 0.87 for nighttime (95% CI 0.85-0.91). For children under 5 (n=524,926), corresponding aOR were 0.87 (95% CI 0.84-0.91) and 0.86 (95% CI 0.83-0.90). Considering a counterfactual in which temperatures never exceeded these thresholds, we estimated 25.8 million (95% CI 14.4-38.2 million) cases of malaria attributable to heat-associated non-use. These results demonstrate the importance of considering climate-behavior interactions for malaria control.

Spatial repellents represent a promising approach to complement existing malaria vector control interventions by reducing contact between mosquitoes and humans. However, the efficacy of passive spatial repellent devices may be affected by environmental conditions, particularly temperature and airflow, which can influence the release of volatile active ingredients. Active-release devices may provide more consistent delivery of spatial repellent compounds. We investigated the efficacy of a commercially available battery-powered spatial repellent device and a low-power solar-powered device designed for potential use in Africa. Laboratory trials were conducted to evaluate the efficacy of the two spatial repellent devices against Anopheles mosquitoes. Protective efficacy was assessed by comparing mosquito probing on human participants during spatial-repellent and control tests. The effect of the devices on mosquito entry into the test chamber was also assessed. The protective efficacy was 86% with the commercial battery-powered device and 80% with the low-power solar-powered device. A Wilcoxon rank-sum test showed that there was no significant difference between the performance of the two devices in terms of protective efficacy (p = 0.856) or entry inhibition (p = 0.7989). The low-power device could be charged using a small, household-level photovoltaic panel, to provide a potentially practical means of delivering spatial repellents in off-grid settings.

Experimental hut trials (EHTs) are WHO-recommended for the entomological evaluation of insecticide-treated nets (ITNs), but several hut designs are in operational use, and structural differences between them may confound efficacy predictions and limit cross-site comparability. We developed a Bayesian hierarchical framework comprising a host-seeking model, which jointly estimates biting deterrence and preprandial mortality while accounting for night-to-night variation and overdispersion, and a postprandial mortality model, which expresses hut and net effects as hazard ratios through a complementary log-log link. We applied it to a comparative trial of four hut designs (East African, West African, Ifakara and Rapley) conducted at a single site in Tanzania, evaluating eight ITNs when new and after twenty washes. Posterior estimates parameterise a vectorial capacity framework to predict reductions in transmission potential. Hut design influenced baseline mosquito behaviour and all three modes of action. Relative to the Rapley reference, baseline feeding rates were substantially lower in the East African and West African huts and closer to Rapley in the Ifakara hut. Preprandial mortality was amplified in the Ifakara hut. Comparing to previous analysis provides evidence that combining mortality before and after feeding into a single endpoint does not reliably reflect impact, supporting the decomposition of entomological outcomes into separate modes of action. Expressing modes of action as mechanism-specific parameters allows the estimates to be carried directly into transmission models. For every net, the predicted reduction in vectorial capacity was greatest in the Ifakara hut and smallest in the West African and Rapley huts. The effect of hut design on predicted impact exceeded that of washing the nets twenty times. Results indicate that the hut design under which trial data were collected should be considered when forecasting population-level effect.

Background In Nigeria, the country with the greatest global malaria burden, funding constraints increasingly require insecticide-treated net (ITN) reprioritization to target those at highest risk. Large language models (LLM)- assisted decision-support tools may facilitate risk-informed ITN planning by supporting malaria programme officers in navigating analyses, interpreting outputs, and translating evidence into operational decisions. We developed ChatMRPT, an LLM-assisted ITN allocation planning tool based on user requirements, and analyzed post-interaction feedback, examined user digital competencies and workplace resources, and identified institutionalization pathways for LLM-assisted intervention planning. Methods A two-phase mixed-methods study began with software requirements gathering workshops (using a prototype) involving representatives from the National Malaria Elimination Programme (NMEP), State Malaria Elimination Programmes (SMEPs), and implementing partners. Phase 2two evaluated ChatMRPT through surveys, guided exercises, and focus group discussions with 34 SMEP officers from 28 Nigerian states. Quantitative data were analyzed using descriptive statistics and profile-based comparisons, while qualitative data were analyzed using reflexive thematic analysis to synthesize user experiences of ChatMRPT and identify institutionalization pathways. Findings Fifty-eight percent (19/33) of participants demonstrated both higher digital competency and adequate workplace resources; the remainder exhibited limitations in one or both domains ([4/33] higher competency/constrained resources; [6/33] higher resources/lower competency). Participants with higher digital competency but constrained workplace resources reported user experiences comparable to those with higher competency and adequate resources, whereas workplace resources alone did not appear to compensate for lower digital competency. Key software requirements included contextual guidance for malaria risk interpretation, operational decision support, and embedded analytical support. Following iterative incorporation of these requirements, ChatMRPT was positively evaluated across participant profiles. Participants viewed institutionalization as dependent on integration into routine malaria planning and adaptability to evolving programme priorities. Interpretation Many malaria programme officers may already have the foundational competency for LLM-assisted decision support. However, there is room to further strengthen digital competencies while facilitating access to basic workplace resources such as stable internet. Institutionalization of LLM tools may depend on addressing these capacity and infrastructural constraints alongside designing explainable, integrated, and flexible systems. Future research should evaluate long-term integration, sustainability, and effectiveness in routine malaria planning. Funding This work was funded by the Bill and Melinda Gates Foundation (INV-036449) and the Center for Health Outcomes and Informatics Research (CHOIR), Loyola University Chicago. The funders had no role in the study design, data analysis, interpretation of findings, or preparation of the manuscript.

Mass drug administration (MDA) using Praziquantel is central to efforts to eliminate Schistosomiasis. However, regions which respond poorly to MDA ("persistent hotspots") have been reported in many regions of Africa, including in Western Kenya. One possible explanation for persistent hotspots is that these areas contain PZQ resistant schistosome parasites. Recent studies have shown that Sm.TRPMPZQ gene is the molecular target for PZQ in schistosome parasites and that mutations in this gene can result in PZQ resistance. This study characterized mutations within Sm.TRPMPZQ in 23,420 miracidia collected from both hotspot and non-hotspot villages in Siaya County, western Kenya. We collected triplicate pools of 780.67 (SD {+/-} 183.47) miracidia from 135 people in five hotspot villages, where S. mansoni prevalence remains high despite over 5 annual treatments, and from 62 people from 5 non-hotspots villages where annual treatment resulted in reduction in prevalence. We extracted DNA from each miracidia pool, amplified 15 amplicons covering 1,695bp of the Sm.TRPMPZQtransmembrane domain and sequenced these to high read depth (21,110x) using a Miseq at KEMRI-CGHR. We identified five high confidence (frequency [&ge;] 0.01) Sm.TRPMPZQ variants. These included four synonymous changes and a non-synonymous variant (p.L1476I). p.L1476I is found at similar frequency in non-hotspot (0.040 {+/-} 0.006) and hotspot villages (0.044 {+/-} 0.0050) (Mann Whitney U=18, p= 0.31) and does not impact PZQ-response in Ca2+ reporter assays. Our studies show that resistance variants in Sm.TRPMPZQ are rare or non-existent in the locations studied and do not explain the existence of hotspots in this region.

All primary healthcare centers owned by the Ugandan government in the Western Region of Uganda were submitted to a questionnaire concerning current availability of POCT from the Essential diagnostic List 2 part 1a and 1b, and the African laboratory inventory done by African Society of Laboratory Medicine and AfricaCDC. The data from the questionnaire was then linked to open source geodata provided by TomTom, and population data to calculate and visualize the accessibility of captured POCT. Findings: Availability of POCT Malaria is almost 100%, HIV 68-90%, and >30% for a majority of the POCT in the EDL-2 panel. 90% of the population in Western Region live within 1 hour by car from most of the essential POCT. Figures in the complementary web-based application visualize the accessibility of POCT for Western Uganda. Diagnostic deserts are visualized. Interpretation: Access to POCT at primary health care facilities in western Uganda has expanded substantially over the past decades. The geo-mapping tool presented here could inform policy decisions on strengthening diagnostic capacity at the national, regional, and provincial level. Funding: Swedish Research Council and Infravis All supplementary materials and a preprint of this submission are available on our OSF repository https://osf.io/j7puk/.

The persistence of Plasmodium vivax is driven by the hidden reservoirs of infection, presenting a key obstacle to elimination. Antibodies persist after asexual infections are cleared from peripheral blood and therefore can indicate current and recent past infections. Here, we present a machine learning algorithm that classifies recent P. vivax infections using serological markers to identify likely hypnozoite carriers. Using serological measurements from year-long observational cohort studies conducted in three low-transmission settings (including negative controls, N=2,635), we selected optimal subsets of markers by balancing sero-diagnostic performance against assay complexity and scalability. We initially trained a random forest classifier and then subsequently we compared several machine learning classifiers. Tree-based methods consistently performed best, although differences were marginal. An online R Shiny application (PvSeroApp) was developed to automate data processing, quality control, and serostatus classification. This algorithm underpins the P. vivax serological testing and treatment (PvSeroTAT) strategy, enabling targeted anti-hypnozoite therapy and strengthening elimination efforts.

BackgroundMalaria control is increasingly challenged by the urban-adapted vector Anopheles stephensi, yet molecular and cellular tools for this species remain scarce, restricting functional genomic studies and the development of genetic control strategies. To help address this gap, we established a new embryo-derived Anopheles stephensi cell line. ResultsWe generated and characterised a novel embryo-derived Anopheles stephensi (SDA-500) cell line capable of sustained growth in vitro. Species identity was confirmed by mitochondrial COI barcoding, and karyotypic analysis revealed a diploid chromosome complement with the presence of a Y chromosome, confirming that at least some cells are of male origin. Transfection conditions were optimized, with TransIT-PRO showing higher efficiency than Lipofectamine-based reagents. Using a dual-luciferase reporter assay, of several promoters tested the Anopheles gambiae polyubiquitin promoter exhibited the strongest and most consistent transcriptional activity in SDA-500 cells. ConclusionsThe SDA-500 cell line provides a stable and genetically validated in vitro platform that supports efficient transgene expression. This resource provides a useful system for functional genomics and molecular manipulation in Anopheles stephensi and is expected to facilitate studies of mosquito biology and contribute to the development of novel malaria control strategies.

Background: Microplastics have been detected in many freshwater ecosystems, including stagnant waters where mosquito larvae develop. Larvae are therefore exposed to microplastic pollution, which may affect their life history traits or microbiota along with immune gene expression. However, these effects have never been tested in mosquitoes, despite their major public health importance as vectors of numerous pathogens. Method: We exposed mosquito larvae, from hatching to adult emergence, to four concentrations of polyethylene microplastics (MPs): 0, 60, 200, and 600 MPs/mL. Fourth-instar larvae and newly emerged adult females were collected for each treatment. To investigate the effects of MPs on gene expression and bacterial microbiota, RNA sequencing and 16S metabarcoding approaches were performed on three biological replicates for each developmental stage. Results: Microplastic exposure induced a non-monotonic dose-dependent transcriptomic response. In larvae, only a limited number of genes were differentially expressed (five to eight per concentration), with immune-related genes downregulated at both low and high concentrations. In adult females, the intermediate concentration (200 MPs/mL) elicited the strongest response with 14 differentially expressed genes (DEGs). Regarding the microbiota, microplastic exposure reduced bacterial diversity in larvae, with the lowest diversity observed at the highest concentration. However, no significant changes were detected in the microbiota of adult females. Conclusion: Overall, this study shows that adult females are affected by larval exposure to MPs (i.e. differential expression in immune-related genes) and warrants further studies in this field. This includes: 1) investigating further the effects of MPs on mosquitoes' populations (e.g. through multi-generational studies), 2) gaining more environmentally relevant knowledge on MP effects (i.e. using MPs with a biofilm and/or adsorbed pollutants) and 3) focusing on the effects of MPs on mosquitoes' vectorial capacities.

Background & objectives: India's fifth National Family Health Survey measured anaemia in all 707 districts, whereas the sixth survey did not. Anaemia is now assessed through a venous blood survey covering 183 districts and reported only at the national level. Using the most recent district-level measurements, we examined whether the remaining survey indicators could predict district anaemia, whether omitting district anaemia altered programme prioritisation, and the value of repeating district-level measurement. Methods: We estimated district anaemia prevalence and uncertainty for children aged 6-59 months and non-pregnant women aged 15-49 years using small-area estimation with design-based variances. We evaluated prediction from the retained survey indicators using both district-level and leave-one-State-out validation, compared district prioritisation under three information scenarios using matched preference draws, and estimated the value of repeating measurement of the same underlying prevalence. Results: Median standard errors of district estimates were 3.57 percentage points for children and 2.22 percentage points for women. The best predictive surrogate had a root mean squared error of 10.14 percentage points for children, of which 9.44 percentage points reflected structural error, representing approximately 2.5 times the root mean squared measurement error. In leave-one-State-out validation, predictions performed worse than the training-set mean. Among the 71 districts prioritised using current estimates, 19.1% were not among the latent top 71 districts. Measuring 183 districts recovered 46.3% of this prioritisation gap when districts were selected according to decision value, compared with 9.6% under equal allocation across States. Interpretation & conclusions: Available survey indicators did not adequately substitute for direct measurement of district anaemia. When measurement resources are limited, the choice of districts to be measured has a greater influence on programme prioritisation than the total number of districts measured, provided differences between measurement platforms are addressed before comparison.

BackgroundResistance of the arbovirus vector Aedes albopictus to pyrethroid insecticides is an emerging threat to vector control programs in Europe. Three knock-down resistance (Kdr) mutations affecting the voltage-gated sodium channels targeted by pyrethroids are known to confer resistance: V1016G, I1532T and F1534C. As these Kdr mutations are actively circulating in Europe, their monitoring is crucial for resistance surveillance programs. However, current Kdr genotyping methods are labor-intensive and costly, limiting their applicability for large-scale high-throughput surveillance. MethodologyNovel digital droplet PCR (ddPCR) TaqMan assays were developed, allowing the quantification of these Kdr mutations from pooled mosquito samples from field populations. The specificity of these assays was validated by using mosquitoes of known genotypes together with synthetic DNA constructs carrying haplotype combinations previously unseen in the field. The assays accuracy was assessed by comparing Kdr frequencies measured from pooled mosquitoes to those derived from individual genotypes. These assays were then used in a pilot surveillance study in mainland France, integrating deltamethrin bioassays, pooled Kdr mutation tracking and vector control interventions data. FindingsThe developed ddPCR assays demonstrated high accuracy and specificity, with matching Kdr mutation frequencies between pooled samples and individual genotyping. The pilot surveillance study confirmed the low prevalence of Kdr V1016G and I1532T mutations in most French populations, though some populations exhibited a moderate decrease in susceptibility to deltamethrin. Deltamethrin spraying intensity was weakly correlated with Kdr I1532T mutation frequency while no correlation was observed with deltamethrin susceptibility, suggesting that insecticide selective pressure from curative vector control activities is at most a minor driver of resistance. ConclusionThese novel ddPCR assays provide a simple, cost-effective and high-throughput method for quantifying the frequency of Kdr mutations in Ae. albopictus populations. Their implementation in routine country-wide surveillance programs will enhance the detection of emerging resistance, and inform vector control strategies, preventing arboviral disease transmission. Author summaryAedes albopictus, the Asian tiger mosquito, is an emerging global threat due to its ability to transmit the dengue, chikungunya and Zika viruses among others. Recurrent use of insecticides to prevent infections selects resistant mosquitoes. Kdr (knock-down resistance) mutations confer resistance to pyrethroid insecticides, like deltamethrin. In order to track the arrival of these mutations in a population, individual PCR tests are routinely used, which is very inefficient and costly. The digital droplet PCR tests presented in this study can instead be performed on pools of mosquitoes from the field, greatly increasing the output while reducing the costs. This makes them ideal for use in the surveillance of these mutations in field populations. We first confirmed that the tests are as efficient in pools as they are on single mosquitoes, then we applied these tests in a pilot study on French field mosquitoes to demonstrate their applicability for use in surveillance. Finally, we explored the link between deltamethrin sprayings, bioassay mortality and Kdr mutations frequency, which is non-existent. This means the current management of mosquito populations via insecticide use is sensible. We believe these tests have a place in streamlining the future surveillance programs of these mutations in the field.