Malaria Journal

The innate immune response of Anopheles mosquitoes is closely associated with the successful development of Plasmodium parasites and their subsequent transmission to humans. However, the extent to which mosquito immune activation correlates with parasite burden during early sporogonic development, and how additional factors may be linked to variation in this early immune response, remains only partly understood. In this study, we examined the immune responses of individual Anopheles coluzzii mosquitoes fed on Plasmodium falciparum infected blood with identical gametocytemia, yet exhibiting different infection intensities at the ookinete stage. We focused on the critical window of ookinete maturation and midgut traversal (12 to 36 hours post-infection), a key phase for parasite establishment, while minimizing confounding factors such as mosquito age, microbiota composition, blood meal variability, and experimental noise. Ookinete burden was estimated in individual mosquitoes by quantifying four well established stage-specific transcripts (ctrp, warp, soap, and cht1), allowing stratification into "low" and "high" ookinete load groups prior to RNA-seq analysis. Transcriptional profiling revealed clear differences between these two conditions, with mosquitoes carrying lower ookinete loads exhibiting stronger upregulation of immune-related genes compared to those with higher burdens, indicating that the reduced parasite establishment during the very early stages of infection is strictly associated with an effective immune activation. Notably, genes involved in L-arginine homeostasis were significantly modulated, highlighting a pathway not extensively explored in the context of mosquito anti-Plasmodium defense. Overall, our findings show that ookinete burden correlates with distinct mosquito transcriptional states during early infection and emphasize the value of ookinete-specific transcripts quantification for dissecting early vector-parasite interactions.

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.

Attractive targeted sugar baits (ATSBs) are a novel vector intervention targeting the sugar feeding behaviour of mosquitos by providing toxic sugar meals. ATSBs have shown strong efficacy in terms of mosquito population reduction in entomological trials but their epidemiological impact remains incompletely understood. This modelling study explores the impact of ATSBs on entomological and epidemiological outcomes over a wide range of transmission settings. We quantify the required killing probabilities to achieve target incidence reductions and demonstrate the sensitivity of ATSB efficacy to mosquito characteristics such as sugar feeding frequency and sac rate. Furthermore, we show that ATSBs can maintain reduced incidence levels previously achieved by long-term insecticide-treated net campaigns. Despite mixed results from clinical trials, these results highlight the high potential of ATSBs as a complementary vector control strategy, while underscoring the critical importance of entomological parameters in predicting public health impact. Realizing this potential will require accurate characterization of mosquito behavior and optimized deployment strategies to ensure sufficient population-level killing probabilities of mosquitoes.

Introduction: The continuing high and inequitable global malaria burden requires improved methods to understand trends and target control. Current methods often involve infection prevalence surveys, but additional parasitological measures could increase precision in estimating infection burdens of communities. Methods: Focusing on the country with most malaria globally, examinations of slides from 10,651 children under five sampled in the 2021 national Malaria Indicator Survey in Nigeria were performed to measure asexual parasite densities in infected children and discriminate sexual stages and species. Variation in these indices among the six different geopolitical zones and 37 individual states including the Federal Capital Territory were compared with the variation indicated by infection prevalence alone. Results: Among the six major geopolitical zones, only one had significantly higher asexual parasite prevalence than others, but parasite densities within infected children revealed more significant variation among the different zones. Comparing results for 2021 with previous data from 2018, one zone showed lower asexual parasite prevalence and two had lower sexual parasite prevalence, but three had higher asexual parasite densities in infected children. Variation in parasite densities among individual states also highlighted significant geographical and temporal differences, extending the detection of heterogeneity within each geopolitical zone. Overall trends were driven by the predominant parasite species Plasmodium falciparum as prevalence and density of the other species were much lower. Conclusions: Findings indicate that understanding complex patterns of geographical and temporal variation is enhanced by including asexual parasite density measurement, providing opportunities for more precise targeting of malaria control within high burden countries.

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.

Background: Angola is one of the most affected countries in sub-Saharan Africa and was off-track to meet the WHO Global Technical Strategy targeting of a 75% reduction in malaria incidence and mortality by 2025. Recent studies highlighted major knowledge gaps regarding malaria vectors in Angola, particularly secondary vectors. A malaria control project initiated in 2007 in Balombo (Benguela Province, central Angola) has monitored malaria dynamics for 18 years, revealing increased Plasmodium prevalence and more frequent detection of non-falciparum species, mainly Plasmodium malariae (in co-infection with P. falciparum) in villagers of the Balombo region. This study aimed to characterize Anopheles populations in this region by assessing species composition, behavioural patterns, and infection rate. And to a less extent, to compare the performance of two indoor and outdoor mosquito trapping methods. Methods: Surveys were conducted during the rainy (December 2023 and 2024) and dry seasons (July 2024) in villages around the town of Balombo. Adult mosquitoes were collected indoors and outdoors using CDC Light Traps and BG-Pro traps in randomly selected houses. Female Anopheles were morphologically and molecularly identified. Primary and secondary vectors were screened for Plasmodium spp. using qPCR-high resolution melting, and positive samples were sequenced. Results: A strong seasonal effect was observed in Anopheles populations, with higher abundance, diversity, and Plasmodium- infection rates during the rainy season. An. funestus remained the main vector, but Plasmodium spp. was also detected in five other species: An. arabiensis, An. gambiae, An. marshallii, An. rufipes, and An. maculipalpis. This is the first report of P. malariae in Anopheles mosquitoes and the first mention of the role of secondary vectors in malaria transmission in Benguela Province. Conclusion: This study provides a thorough and updated inventory of Anopheles species in the Balombo region. Molecular biology confirmed the presence of P. falciparum and P. malariae in several species of Anopheles underscoring the existence of a complex transmission system in villages of Balombo. Infected exophagic and exophilic Anopheles species, exhibiting opportunistic behaviour, likely contribute, as secondary vectors, to the increase of Plasmodiumspp. transmission in the study sites. Therefore, confirming the need to implement complementary interventions targeting outdoor malaria transmission.

BackgroundAntimalarial drug efficacy is threatened by the development of drug resistance. In Rwanda, high levels of resistance have been reported in the past, including mutations in the Pfk13 gene associated with partial resistance to artemisinin, leading to delayed parasite clearance of Artemether-Lumefantrine (AL). Therefore, continuous molecular surveillance of drug resistance markers is required. Here, we report polymorphisms in the Pfcrt, Pfdhps, and Pfdhfr genes in samples collected in Rwanda in 2018. MethodsSamples from three sites (Masaka, Rukara, and Bugarama) were collected during a therapeutic efficacy study (TES) conducted in 2018. Targeted amplification of resistance genes of interest was performed by PCR, and products were sequenced by Sanger sequencing. ResultsThe K76T Pfcrt mutation was found in 10% of the samples, only in Bugarama. For Pfdhps, the A437G, K540E and A581G mutations were detected in 94%, 94% and 55% of the samples, respectively. For Pfdhfr, the N51I, S198N, C59R and I164L mutations were found in 99%, 99%, 88% and 16% of samples, respectively. The latter was only detected in Masaka and Rukara. ConclusionsWe confirm the slow and partial recovery of chloroquine susceptibility in the country. All previously reported Pfk13 mutants were Pfcrt wild type, thus possibly selected by AL. The prevalence of Pfdhps and Pfdhfr mutants, including those with the highly resistant I164L mutation, is high and increasing, despite the absence of drug pressure. It is imperative to closely monitor resistance and efficacy of AL and other treatment options as part of the malaria surveillance program and resistance mitigation strategy in the country.

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.

Background: Invasive non-typhoidal Salmonella (iNTS) disease causes high childhood mortality in sub-Saharan Africa. Malaria infection is postulated to increase iNTS risk through impairment of bactericidal immunity. We quantify malaria's causal contribution to iNTS and explore burden mitigation via integrated antimalarial and iNTS prevention strategies. Methods: We analyzed 443 weeks of surveillance data (September 2017-March 2026) from Kisantu health zone, Democratic Republic of Congo. Analyses included interrupted time series of perennial malaria chemoprophylaxis (PMC; November 2023) and R21/Matrix-M vaccine (October 2024) rollouts, causal mediation analysis, calibration of an age-structured co-infection Susceptible-Infected-Recovered-Susceptible (SIRS) model via the neural ordinary differential equation (ODE) adjoint method, and a 15,435-scenario grid search for NTS infection elimination coverage thresholds. Findings: Malaria burden was 80% lower at PMC full coverage (incidence rate ratio [IRR] 0{middle dot}197, 95% CI: 0{middle dot}096-0{middle dot}401 ; p<0{middle dot}001). A declining iNTS trend emerged post-ramp (IRR 0{middle dot}990/day, 95% CI: 0{middle dot}981-0{middle dot}998 ; p=0{middle dot}018). The calibrated SIRS model estimated ~12-fold iNTS susceptibility during malaria infection ({psi}M[-&gt;]N = 12{middle dot}0, 95% CI: 3{middle dot}6-28{middle dot}3). Combined malaria prevention strategies provided immediate iNTS burden reduction ([&le;] 37%), but full iNTS elimination requires a [&ge;]70% efficacious iNTS vaccine. Interpretation: Malaria control interventions deliver meaningful iNTS co-benefits. This analysis support their prioritization along with further evaluation of integrated malaria-iNTS prevention strategies in high-burden settings. Our results show that full NTS elimination additionally requires a high-efficacy iNTS-specific vaccine. Funding: The Gates Foundation (INV-047715, INV-007844, INV-082265, INV-077454), The European and Developing Countries Clinical Trials Partnership (RIA2017S-2027), and the Swedish Styrelsen for internationellt utvecklingssamarbete (17106)

Background: Severe malaria remains a major cause of hospitalization in malaria-endemic sub-Saharan Africa, and anemia is among its most frequent severity criteria. Data on the impact of anemia on hospital length of stay (LOS) among adults with severe malaria remain limited in Central Africa. We assessed the impact of anemia on LOS among adults hospitalized for severe malaria in Brazzaville, Republic of Congo. Methods: We conducted a cross-sectional, descriptive, and analytic study of medical records from adult patients ([&ge;]18 years) diagnosed with severe malaria (WHO criteria) and admitted to four hospitals in Brazzaville between January 2020 and December 2024. We dichotomized LOS as short (<5 days) or prolonged ([&ge;]5 days). Multivariable logistic regression, using a backward stepwise procedure, identified factors independently associated with prolonged LOS; isolated hemoglobinuria served as the reference severity category. Results: Among 523 patients hospitalized for severe malaria, 296 met the inclusion criteria. Median age was 25.5 years (IQR 20-40); 51.7% were female, and 82.1% had a low socioeconomic status. Median hemoglobin was 10.55 g/dL, and median LOS was 4 days (IQR 3-5). In multivariable analysis, prolonged LOS was independently associated with delay to consultation (aOR 1.25/day, 95% CI 1.09-1.44), isolated anemia (aOR 7.07, 1.23-40.55), convulsions (aOR 5.86, 1.81-18.97), prostration (aOR 4.42, 1.21-16.2), and impaired consciousness (aOR 4.29, 1.17-15.7). The strongest associations involved combined severity criteria: anemia with prostration (aOR 8.58), anemia with jaundice or hemoglobinuria (aOR 16.21), and impaired consciousness with anemia (aOR 33.31). Conclusions: Anemia is a major, potentially modifiable determinant of prolonged hospitalization in adults with severe malaria, with a markedly amplified effect when combined with neurologic impairment. Early anemia screening, improved access to safe blood transfusion, and reduced delays to care could shorten hospital stays and ease the burden on health systems in this setting.

Malaria parasite virulence will be impacted by naturally occurring variation in multiplication rates. Clinical isolates of Plasmodium falciparum exhibit a range of multiplication rates under exponential growth conditions in culture, but it needs to be discovered if relative multiplication rates under competitive conditions are principally defined by this underlying multiplication rate potential. Relative multiplication rates of P. falciparum lines were investigated in 14-day competition assays against a standard competitor clone HB3, with other long-term laboratory-adapted clones showing similar or higher mean per-48-hour rates ranging from 0.98 to 1.51 relative to HB3. In contrast, thirteen Ghanaian clinical isolates cultured for close to two months prior to assay had lower rates, ranging from 0.46 to 0.76 relative to HB3. Analysing the single-genotype clinical isolates and laboratory-adapted clones, there was a highly significant correlation between the competitive rates and previously determined exponential rates (Spearmans rho = 0.95, P = 0.0003), which accounted for most of the observed variance (Pearsons r2 = 0.77, P = 0.004). Testing the effect of alternative nutritional supplementation with human serum or Albumax showed a majority of parasites had consistent competitive rates under either condition, indicating that intrinsic differences determine most variation among parasites.

Artemisinin partial resistance (ART R) associated Plasmodium falciparum Kelch13 mutations is expanding across Africa. However, their population level consequences remain poorly understood, particularly in settings of moderate to high malaria transmission. We investigated associations between Kelch13 and partner drug mutation prevalence and monthly confirmed malaria incidence across 16 Ugandan districts from 2017 to 2022, using integrated molecular, epidemiological, climatic, malaria control, and antimalarial resistance data and negative binomial regression. Combined prevalence of WHO validated and candidate Kelch13 mutations increased over the study period. Each 10 percentage point increase in Kelch13 prevalence was associated with a 13.1% higher incidence after multivariable adjustment (aIRR 1.131, 95% CI 1.090 to 1.174). Furthermore, each 10% increase in pfcrt K76T was associated with a 24% increase in malaria transmission. As expected, ITN distribution >80%, IRS coverage >90%, and higher altitude were associated with lower incidence, whereas temperatures of 25 to 30C, relative humidity of 60 to 80%, and higher precipitation were associated with higher incidence. The persistence of the Kelch13 and pfcrt K76T association after adjustment suggests that expanding ART R associated parasite populations are independently associated with malaria incidence. The relationship may be bidirectional. These findings support interpreting ART R within its ecological and intervention context and integrating molecular, epidemiological, and malaria control surveillance to safeguard antimalarial efficacy.

Immunity against Plasmodium falciparum malaria is acquired slowly and incompletely through repeated infections, leading to differing levels of protection among individuals based on their history of exposure to the disease. Since malaria is a climate sensitive vector-borne disease, immunological history is shaped by the local climate. Nonetheless, compartmental malaria models, which may be used to inform policy, implicitly represent immunity as a population-average trait --- an assumption that can lead to incorrect estimates of clinical prevalence by age, and of the overall burden of malaria. As the rate at which immunity is acquired depends on transmission intensity, and therefore on background climatic conditions, the error introduced by this assumption is expected to be climate-sensitive. We quantify how individual exposure histories shape community-level prevalence, and identify the age groups for which resolving immunity individually matters most across climatic regimes. For this, we introduce VECTRI-ABM, a grid-based framework coupling the climate-driven vector component of the VECTRI model to an agent-based representation of the human population, resolving immunity at the individual level. The framework was calibrated against entomological and epidemiological observations from Dielmo and Ndiop (Senegal, 1990--2003) and evaluated against reported entomological inoculation rates, Malaria Atlas Project estimates of parasite rate in children aged 2--10, and confirmed cases from Senegal's National Malaria Control Program (PNLP). We then applied it across Senegal and The Gambia, which encompass a pronounced Sahelian-to-tropical rainfall gradient, and compared simulations in which immunity develops individually against a counterfactual where agents carry the local population-average immunity, thus mimicking the assumption made by standard models. Results show that the averaging approach misallocates burden across age, underestimating clinical prevalence in children, especially so in the wettest high-transmission regions, and overestimating adult asymptomatic reservoirs, with implications for transmission-targeting interventions. We find that the simpler approach to immunity is adequate for immunologically mature populations, typically above 15 years of age, and that the error it introduces elsewhere is climate-dependent.

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.

The effectiveness of malaria vector control is increasingly threatened by widespread insecticide resistance, particularly to pyrethroids. In response, the World Health Organization recommends dual-active ingredients (dual-A.I.) insecticide-treated nets (ITNs) in resistant settings. This study evaluated the efficacy of several dual-A.I. ITNs in two experimental hut trial sites an area with high insecticide resistant malaria vectors in Tanzania with differing vector species composition and bionomics. Five ITNs were evaluated: a pyrethroid-only net (MAGNet(R)), three pyrethroid-piperonyl butoxide (PBO) nets (OlysetTM Plus, Veeralin(R), and PermaNet(R) 3.0), one dual-A.I. net (Interceptor(R) G2; chlorfenapyr + alpha-cypermethrin), and an untreated control net. Mosquitoes were collected from huts, morphologically identified, and monitored for mortality up to 72 h. WHO cone and tunnel tests using susceptible An. gambiae s.s. were conducted for quality control, while field-collected mosquitoes were used for bio-efficacy monitoring. Molecular species identification was performed using PCR assays, and genomic DNA was extracted from An. funestus s.l. and An. gambiae s.l. collected from the trials. All ITNs met WHO bio-efficacy criteria in cone and tunnel tests. An. gambiae s.l. predominated in Mwagagala (77%) but was rare in Magu (4%), where An. funestus complex was dominant. In Magu, Interceptor(R) G2 and Veeralin(R) achieved significantly higher mortality than MAGNet(R) at 24 h (53% and 45% vs 24%) and 72 h (61% and 53% vs 24%). In contrast, no significant mortality differences were observed among ITNs in Mwagagala. High resistance intensity was detected in both vector groups (mortality <20% in bioassays). In An. funestus from Magu, the 4.3 kb-SV resistant genotype was fixed, while G454A-CYP9K1 resistance was highly prevalent (RR: 0.70-1.00). Interceptor(R) G2 and Veeralin(R) outperformed the pyrethroid-only reference net against An. funestus complex but not against predominantly An. arabiensis populations.

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.

Genomic tools can support malaria control policy through surveillance of Plasmodium falciparum populations, tracking antimalarial drug resistance, pfhrp2/3 deletions that compromise rapid diagnostic tests, and selection at the circumsporozoite protein (PfCSP) vaccine target, as well as through molecular correction of therapeutic efficacy studies (TES). Multiplex Amplicons for Drug, Diagnostic, Diversity, and Differentiation Haplotypes using Targeted Resequencing (MAD4HatTeR), a comprehensive amplicon sequencing panel covering up to 276 targets, supports these applications but is tailored to research rather than routine programmatic use. We developed P. falciparum Public Health Amplicon Sequencing Tool (PfPHAST), a 56-target derivative of MAD4HatTeR spanning drug resistance loci, pfhrp2/3 deletion, PfCSP genotyping, non-falciparum species identification, and 20 high-heterozygosity microhaplotype loci for TES classification. We compared PfPHAST and MAD4HatTeR using laboratory strain controls, including two-strain dilution series and a five-strain mixture, across parasite densities of 100 to 10,000 parasites/L. At matched per-target depth, PfPHAST achieved a higher quality-control pass rate than MAD4HatTeR (94.4% versus 90.0%) and distributed reads more evenly across targets. The panels showed comparable recall and precision for drug resistance codons and microhaplotypes, reaching near-complete recall above 40% within-sample allele frequency (WSAF) at all densities, with reduced sensitivity for minor alleles below 10% WSAF at low parasite density in both panels. Observed and expected WSAF correlated strongly for both panels, and both resolved a five-strain polyclonal mixture, including a 5% minor strain. By concentrating sequencing capacity on targets of greatest programmatic relevance, PfPHAST offers a scalable, lower-cost alternative to comprehensive research panels without sacrificing performance on shared targets, complementing MAD4HatTeR for routine molecular malaria surveillance.

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.