Model reduction and analysis: A case study of a malaria control model.

Chemoprophylaxis (in travellers) and seasonal chemoprophylaxis and preventive treatment (in endemic areas) are important but not exclusive elements of malaria prevention, which hinges on vector repelling in travellers and more comprehensive vector control measures in endemic areas. In malaria-endemic countries, a comprehensive strategy combining drug-based prevention, vaccines and vector control is essential to reduce disease burden. Chemoprophylaxis in endemic settings primarily involves intermittent preventive treatment in vulnerable groups and mass drug administration in communities, rather than continuous daily chemoprophylaxis (as recommended for non-immune travellers). Vaccination has become a groundbreaking addition to the malaria control portfolio in endemic countries. The World Health Organization-endorsed RTS,S/AS01 (Mosquirix®) and R21/Matrix-M vaccines rolled out in many highly malaria-endemic sub-Saharan African countries, in addition to other malaria control tools, provide overall moderate protection in young children but significantly reduce severe disease and death, and are considered safe. For travellers, malaria vaccines are not available to date. Effort is being put into the development of monoclonal antibodies against malaria as a preventive treatment strategy both to provide protection for the immunocompromised or unvaccinated high-risk individuals before exposure and to disrupt seasonal malaria transmission with a single application. The evolution of malaria preventive tools is a dynamic process, with numerous novel developments on the horizon. For travel medicine indications, further harmonisation of recommendations on the one hand, but alsomore sophisticated personalisation of recommendations, is envisaged.

Status of pfcrt and pfmdr1 mutations in Plasmodium falciparum isolates in Gabon in 2019 after chloroquine withdrawal.

Despite significant resources invested in malaria control interventions for several years, the disease remains a persistent health challenge impacting millions of people especially children and pregnant women in the lower income regions of the world. Progress towards eliminating malaria has stagnated, in part due to the limited consideration of the social determinants of the disease such as poverty. We conducted a qualitative study exploring stakeholder perspectives on social determinants of malaria including lived experiences with the disease in Wakiso, one of the districts with high malaria cases in Uganda. This was a cross-sectional study using fourteen key informant interviews at community, health facility, district and national levels, 10 focus group discussions among community members and community health workers, and 11 in-depth interviews with household heads. Data collection was conducted in English and Luganda (local language), transcribed, coded using Atlas.ti. (version 6) and Nvivo (version 14) and analysed using a thematic approach. This paper focuses on the perspectives on poverty as a social determinant of malaria. The participants linked poverty to malaria through five themes: poor housing conditions; limited access to malaria control services; limited water and sanitation-related exposures; occupational and psychological exposures; and heavy economic burden (consequences) of malaria. The themes are presented under various sub-themes which separately highlight in detail the different pathways of the malaria-poverty relationship. These include: substandard housing structures enabling mosquito exposure; building close to mosquito breeding grounds; homelessness and street life; inadequate malaria prevention and treatment services; limited access to water supply; poor sanitation and waste management practices; occupational exposures; social and psychological constraints; high treatment costs; undernutrition causing low immunity; and loss of income. These perspectives emphasize a cyclic poverty-malaria relationship through the high treatment costs and loss of income due to illness or livelihood disruption. Malaria is rooted in economic inequalities suggesting that improvement in malaria control interventions through policies and frameworks that address poverty as a consequence, and driver of the disease should be prioritised. The study underscores the need for integrated malaria control strategies that address vulnerabilities related to poverty in all its dimensions-economic, environmental and psychosocial.

A high-resolution molecular tool for tracking and differentiating closely-related Plasmodium vivax populations is critically needed. This study aimed to develop and validate a novel single nucleotide polymorphism (SNP) barcode to monitor the progress of malaria elimination in the Great Mekong Subregion (GMS). A total of 210 P. vivax clinical samples were collected across four time points in three international border areas: China-Myanmar border, Thailand-Myanmar border, and Bangladesh-Myanmar border. Parasites were genotyped at 36 SNPs using MassARRAY technology (Sequenom), with Sanger sequencing validation for low-efficiency loci. The complexity of infection (COI) was estimated via a maximum likelihood approach implemented in COIL, while genetic diversity metrics were computed in GenAIEx version 6.5. Population differentiation was assessed through molecular variance analysis, Mantel rank test, and pairwise FST estimation. Genetic structure was resolved using principal component analysis, phylogenetic analysis, and ADMIXTURE. 198 samples were successfully genotyped at 22 validated SNPs, revealing 37.9% polyclonal infections. The proportion of polyclonal infections differed significantly among the five P. vivax populations (P = 0.0001, Pearson Chi-square test, χ2 = 23.15), with 2020 CMB samples having the highest proportion (56.1%). The average COI was highest in BMB parasites (1.109 ± 0.007). The TMB 2018 samples exhibited the maximal nucleotide diversity (π = 0.342 ± 0.033) and expected heterozygosity (He = 0.325 ± 0.04). The P. vivax populations from the western GMS showed significantly reduced genetic diversity in recent years compared to earlier timepoints (0.372 ± 0.009 vs. 0.426 ± 0.009; P < 0.0001, Student's t-test). Pairwise FST values indicated moderate to high genetic differentiation (0.165 - 0.417) across nine population pairs, except for the temporally proximal CMB populations, which showed low differentiation. Structure analysis consistently resolved three discrete genetic clusters corresponding to CMB, TMB, and BMB parasite populations. This 22-SNP barcode provides a high-resolution genotyping tool capable of differentiating P. vivax parasite infections from the western GMS. Our data demonstrate that sustained malaria control interventions drive the fragmentation of P. vivax populations into genetically distinct transmission foci, creating opportunities for elimination strategies in border hotspots.

Adherence to radical cure for Plasmodium vivax remains a major challenge for malaria control programmes. Novel regimens, such as 7-day high-dose primaquine regimen and single-dose tafenoquine, may improve treatment adherence and antirelapse effectiveness but can increase the risk of haemolysis in individuals with glucose-6-phosphate dehydrogenase deficiency. Stakeholder (ie, policymakers, providers, patients, etc) perceptions of these risks may significantly influence the acceptability and uptake of these regimens. Understanding these perceptions is essential for policymakers to design effective communication and implementation strategies of novel radical cure strategies. Guided by qualitative methodology and a risk perception lens, this study explored P. vivax malaria stakeholders' experiences and interpretations of novel radical cure regimens. Between February and September 2023, 58 semistructured interviews and 6 focus group discussions were conducted at Arba Minch General Hospital and three health centres and health posts within the Gamo Zone of the South Ethiopia Regional State: Lante, Shecha and Shele. Participants included clinical trial staff and participants, health centre personnel, health extension workers and routine care patients. The 7-day-high-dose primaquine regimen and single-dose tafenoquine were viewed as promising solutions to the adherence challenges of the standard 14-day primaquine regimen; however, participants raised concerns about the effectiveness of the shortened treatment duration and the safety of the increased daily dose of primaquine. A risk perception lens revealed that concerns about effectiveness and safety were influenced by prior public health messaging emphasising completion of the full 14-day regimen alongside fears of overdosing and drug-induced haemolysis. Pill characteristics, including number, shape and colour as well as fears of overdosing and drug-induced haemolysis also contributed to apprehension about the safety of these regimens. Participants' perceptions of shortened radical cure regimens were shaped by concerns about effectiveness and safety, influenced by prior treatment messaging and pill characteristics such as higher dosing and pill burden. A risk perception lens can inform implementation and communication strategies for novel regimens, where addressing user perceptions alongside practical improvements, such as reducing pill burden, is essential for optimising uptake and adherence.

Insecticide resistance in Anopheles mosquitoes threatens the effectiveness of key malaria control tools such as insecticide-treated nets (ITNs) and indoor residual spraying (IRS) in Ethiopia. Genomic analysis is essential to model known and novel molecular markers of insecticide resistance for effective resistance management. This study investigated insecticide resistance genes using whole-genome sequencing in a major malaria vector, Anopheles arabiensis sampled across the whole regions of Ethiopia and found high geographic and temporal variability in genes associated with insecticide resistance. The Vgsc-L995F target-site substitution in the voltage-gated sodium channel gene was highly prevalent in northern Ethiopia but less common at other sites. Metabolic modes of resistance in western Ethiopia were indicated by the high frequencies of copy number variants observed at the cytochrome P450 cluster Cyp6aa/p and the carboxylesterase Coeae2-7g. Frequencies of genetic markers associated with molecular target sites and metabolic resistance were generally lower in the Central Rift Valley. However, copy number variants (CNVs) at Gste2 and Cyp9k1 were observed at high frequency. We observed seasonal shifts in both target-site and metabolic marker frequencies, including increasing frequencies of Vgsc-L995F and several cytochrome P450 variants during the major transmission season. These patterns were specific to each location. Findings indicate that molecular insecticide resistance arises from a complex interplay of factors, including malaria control interventions, agricultural practices, human behavior, and possibly vector behavior. Selection scans revealed signals of selection on chromosome 2L, centered on the Coejhe1-5e genes in Werkamba, northernmost Ethiopia. Additional signals were detected on chromosome 3L (~ 20Mb), near genes that may regulate detoxification pathways, including those associated with the ubiquitin-proteasome system in Asossa, western Ethiopia. Findings highlight the importance of integrating genomic surveillance of resistance markers into entomological monitoring to strengthen insecticide resistance management. They also underscore the need to investigate lesser-known sources of adaptive change that may have significant consequences for vector control.

Because P. falciparum and P. vivax coexist in the Gedeo Zone, accurate malaria diagnosis is essential for effective case management. Low parasitemia and pfhrp2/3 gene deletions limit the performance of conventional HRP2-based RDTs. The new Bioline Malaria multi-Ag Pf/Pf/Pv RDT, detecting HRP2 and dual pLDH for both species, addresses this limitation. This study evaluates its field performance in detecting low-density infections during peak transmission season using expert microscopy and qPCR as references. A health facility-based cross-sectional study enrolled 579 febrile patients from health facilities in Gedeo Zone during the major transmission period from October to December 2025. Venous blood was tested with the conventional and multi-antigen Bioline RDTs per manufacturer directions, alongside expert microscopy and qPCR. Diagnostic performance parameters were calculated, comparing RDTs to expert microscopy and qPCR. Among 579 febrile patients, malaria prevalence was 36.4% (211/579) by light microscopy, 33.2% (192/579) by multi-antigen RDT, and highest with qPCR 40.8% (236/579), which also detected a greater number of P. falciparum (20.7%), P. vivax (16.9%), and mixed infections (3.3%). Using microscopy as a reference, multi antigen RDT showed high sensitivity 86.2% (95% CI: 80.8-90.6%) and specificity 98.6% (95% CI: 96.8-99.5%) with almost perfect agreement (κ = 0.87); however, against qPCR, sensitivity decreased for P. falciparum 77.1% (95% CI: 71.2-82.3%) and despite consistently high specificity 97.1% (95% CI: 94.7-98.5%), indicating missed submicroscopic infections. The Bioline Malaria Ag Pf/Pf/Pv RDT demonstrated high specificity and substantial agreement with microscopy but moderate sensitivity compared with qPCR, suggesting that low-density infections were overlooked. These findings highlight the need for more sensitive diagnostic techniques for malaria surveillance and control in endemic environments while supporting the clinical utility of RDTs for regular clinical diagnosis.

Alternative vector control tools are needed to address rising malaria burden in Papua New Guinea (PNG), where outdoor transmission and reduced insecticidal efficacy of insecticide-treated nets (ITNs) create persistent protection gaps. Volatile pyrethroid spatial emanators (SEs) release active ingredients that deter or kill mosquitoes, and 2 SE products have recently been WHO prequalified for malaria vector control. We evaluated the entomological efficacy of SumiOne™ SEs in 2 villages in PNG. Spatial emanators were installed indoors only or both indoors and outdoors in semi-enclosed peridomestic spaces, with 12 houses per intervention arm. Human landing catches and sentinel cage bioassays were conducted over 8 weeks and compared with a control arm. Human-vector contact was significantly reduced indoors, with a protective efficacy of 60%-80% in both intervention arms for 5-6 weeks, declining at approximately 6%-7% per week. No significant reduction in outdoor human-vector contact was detected. Indoor sentinel cage bioassays showed >80% 24-hour mortality for 7 weeks, and complete 60 minute knockdown and inhibition of probing for 8 weeks. SumiOne™ SEs substantially reduced indoor mosquito exposure and may complement ITNs for malaria control in PNG. Future research should prioritize increased longevity and stronger outdoor impact.

Changes in vector biology and behaviour resulting from interventions such as indoor residual spraying (IRS), long-lasting insecticidal nets (LLINs), and the use of insecticidal aerosol sprays, coils, and other mosquito control measures underscore the need for strengthened and regular vector surveillance. Understanding vector behaviour and species diversity is crucial for adapting interventions to achieve more effective and sustainable malaria control. This study examined malaria vector biting behaviour and transmission risk in Simiw, a community located in the coastal forest of Ghana's Central Region. Adult malaria vectors were collected using human landing catch (HLC) method during the dry and rainy season of 2021. Vector densities, biting patterns and entomological transmission indices of An. gambiae s.l. were evaluated. Molecular techniques were employed to identify vector species, detect Plasmodium falciparum infection, and analyze insecticide resistance genes. A total of 4,173 mosquitoes from four different genera were collected during the sampling period, with a surprising higher abundance recorded in the dry season compared to the rainy season. Indoor collections of An. gambiae s.l. outnumbered outdoor collections in both seasons. An. coluzzii was the most prevalent species, making up 79.0% (678/858), followed by An. gambiae s.s. at 17.6%, hybrids of An. gambiae and An. coluzzii at 1.9% (16/858), and An. melas at 1.5% (13/858). The biting rate for An. gambiae s.l. peaked in the early morning hours between 04:00 and 05:00. The overall entomological inoculation rate (EIR) was calculated at 61.20 infective bites per person per year. The study found higher abundance and biting rates of An. gambiae s.l. during the dry season, with both the HBR and EIR being greater indoors than outdoors. The observed species diversity and transmission risk indices emphasize the importance of ongoing vector monitoring.

Malaria remains a major health burden in South America, but the extent to which climate change will reshape vector communities is poorly understood. We modeled the distributions of 15 key Anopheles species, representing primary, secondary, and unclassified vectors, under current and future climate scenarios (SSP2-4.5, SSP5-8.5) using ensemble ecological niche models calibrated with 20 climatic and physiographic predictors. We quantified niche breadth and overlap and achieved high predictive performance. Analyses in environmental space revealed moderate overlap between primary and secondary vectors and lower overlap between primary and unclassified vectors, although ecological breadth did not differ significantly among classes. Future projections revealed divergent trajectories among Anopheles species. Climate-sensitive primary vectors such as An. darlingi is projected to undergo severe contractions and fragmentation, particularly within the Amazon Basin, whereas An. albimanus and An. aquasalis are predicted to expand southward under SSP5-8.5. Several secondary or unclassified species (An. janconnae, An. marajoara, An. argyritarsis) also showed potential for expansion. These results suggest a major reorganization of vector assemblages, with potential epidemiological implications. Understanding this reshuffling and clarifying the roles of emerging vectors will be crucial for guiding future malaria control and surveillance strategies in South America.

Major advances in Plasmodium sequencing approaches, bioinformatic pipelines, and data analysis tools have provided valuable insights into malaria epidemiology from parasite genomic data. However, translating genetic data into actionable information for decision-makers remains a challenge. Significant barriers limit the integration of these advances into a functional data analysis ecosystem that produces standardized, interpretable results for use by national malaria control programs. The Plasmodium Genomic Epidemiology network convened 18 subject matter experts across 15 institutions at the Reproducibility, Accessibility, Documentation, and Interoperability Standards Hackathon in 2023 to identify available analysis tools, evaluate software standards, improve documentation, and outline workflows. Eight use cases for genomic data were identified, and a subset was developed into analysis workflows comprising a series of connected functionalities. Software tools were then mapped against functionalities to outline a modular approach to data analysis for these use cases. In addition to outlining workflows, a set of objective criteria was developed for evaluating software standards. A total of 40 Plasmodium genomic analysis tools were identified, 22 of which were prioritized for software standards evaluation. Additional tutorials were developed for 10 tools in the form of reproducible code applied to shared datasets. These resources are available on PGEforge (mrc-ide.github.io/PGEforge), a new community resource that serves as a central, open repository for current and future resources for malaria genomic data analysis.

This study aimed to assess the prevalence and risk factors of molecular markers of Plasmodium (P.) falciparum resistance to sulfadoxine-pyrimethamine (SP) among pregnant women in Bobo-Dioulasso, and to examine their association with maternal anaemia. This cross-sectional study was conducted between October and December 2022 among 288 SP-naïve pregnant women attending antenatal care at the Centre Médical Urbain of Lafiabougou. Dried blood spots were collected, and P. falciparum infection was detected using quantitative polymerase chain reaction (qPCR) targeting the varATS gene. Positive samples were genotyped for mutations in the Pfdhfr and Pfdhps genes using a nested PCR approach followed by restriction fragment length polymorphism analysis. Multivariable logistic regression models were used to identify predictors of resistance markers and maternal anaemia. Among the 172 qPCR-confirmed P. falciparum–positive samples, the Pfdhfr triple mutant haplotype (N51I, C59R, S108N) was detected in 33.7% of cases, while the Pfdhps A437G mutation was present in 82.7%. No Pfdhfr I164L or Pfdhps K540E mutations were observed. Gestational age was independently associated with carriage of the triple Pfdhfr mutation (adjusted OR = 2.2, 95% CI: 1.1–4.5). Both gestational age (adjusted OR = 2.5, 95% CI: 1.2–4.9) and infection with parasites carrying the triple Pfdhfr mutation (adjusted OR = 3.9, 95% CI: 1.8–8.3) were significant predictors of maternal anaemia. The relatively high prevalence of SP resistance markers indicates sustained drug pressure in this setting. Although SP appears to remain effective for intermittent preventive treatment in pregnancy, continued molecular surveillance is warranted to inform malaria control policies.

Malaria remains a major global health burden. Vaccines targeting the Circumsporozoite protein represent a significant advancement in malaria control and have demonstrated meaningful public health impact. However, no validated correlate of protection has yet been established, making it critical to assess antibody functionality alongside antigen-specific antibody quantitation to evaluate vaccine candidates. Antibody-dependent phagocytosis is one such mechanism that contributes to parasite clearance, which can be modelled using cell lines. While THP-1 (monocyte-like) assays have been described, HL-60 (neutrophil-like) models are less established in the malaria field. Here, we aimed to develop and characterize a PfCSP repeat region (NANP6) bead-based opsonophagocytic assay using differentiated HL-60 cells, to adapt a THP-1 assay, and to assess IgG subclass- and FcγR-dependent differences between these in vitro models. Ultimately, our goal is to demonstrate the potential application of these assays for exploratory assessment of human clinical samples. Fluorescent NANP6-coated beads were opsonized with recombinant IgG1-2-3-4 monoclonalantibodies (identical binding domain, different Fc) or human sera from an RTS, S/AS01E trial. Opsonized beads were incubated with model effector cells, phagocytosis was quantified by flow cytometry, and EC50 values were derived using four-parameter logistic regression. FcγR-blocking experiments and assay performance (precision, dilution linearity, sensitivity) were evaluated. Both assays discriminated phagocytic potency across IgG subclasses, revealing that FcγR dependence differs by subclass and between the two cell models. Both assays were successfully adapted to human sera, demonstrating robust performance with high precision, strong dilution linearity, and high sensitivity across samples with varying anti-CSP antibody levels. These characteristics support their suitability for reproducible assessment of functional antibody responses in clinical samples. Two opsonophagocytic assays recapitulating monocyte-like and neutrophil-like effector pathways were developed and characterized. Both demonstrated precise, sensitive, and quantitative measurement of antibody-dependent phagocytosis in human sera, while capturing distinct functional differences between the models, supporting their use as exploratory tools to evaluate functional antibody responses in clinical samples.

The post-2000 period in South America presents a striking divergence in malaria epidemiology: Ecuador achieved a >99% reduction in malaria incidence approaching elimination, while Venezuela experienced one of the most severe resurgences in modern public health history. We examined the association between governance factors, health policies, intersectoral coordination, and malaria control outcomes by comparing the national experiences of Ecuador and Venezuela. We conducted a comparative ecological study using epidemiological data from WHO World Malaria Reports and PAHO updates (2000-2023), integrated with political stability indices, health expenditure data, GDP per capita, poverty rates, and crude-oil price series from World Bank and CFR databases. A joinpoint (segmented) regression model was applied to each country's log-transformed malaria incidence time series to identify statistically significant inflection points and estimate annual percentage change (APC) per segment. A multi-dimensional framework assessed governance, health-system capacity, surveillance infrastructure, intersectoral coordination, and community engagement. We also examined systemic fragility through concurrent infectious disease trends (diphtheria, measles, tuberculosis) in Venezuela. Use of AI language models is declared in the Methods. Ecuador reduced cases from 106,641 (2001) to 757 (2023), a 99.3% reduction. The joinpoint model identified two significant inflection points (2008, 2014) delineating three phases of declining trend (APC: , , ). Venezuela experienced a 1,217% increase from 35,500 (2000) to a peak of 527,627 (2018); a single joinpoint at 2014 marked a shift from non-significant endemic transmission (APC: , ) to explosive resurgence (APC: , ), temporally coinciding with the global crude-oil price collapse and ensuing fiscal contraction. Post-2018 case declines in Venezuela are accompanied by concurrent surveillance collapse, mass emigration, and reduced care-seeking, suggesting underreporting rather than epidemiological improvement. Pearson correlations with malaria incidence were observed for political stability index (), health expenditure (% GDP, ), poverty rate (), and crude-oil price () in Venezuela. These correlations should be interpreted with caution given potential confounding and collinearity among indicators. Comparison across five governance-resilience domains using PAHO/WHO operational criteria shows systematic differences between countries. Resurgence of diphtheria (>1,600 cases), measles (>7,300 cases), and tuberculosis (near-doubling of incidence) in Venezuela occurred concurrently with the malaria resurgence. Health-system resilience and governance quality appear to be associated with the relationship between macroeconomic shocks and epidemiological outcomes. The strong collinearity between political stability and commodity-price cycles in Venezuela precludes definitive attribution to either factor independently. Technical interventions may be insufficient without stable governance, protected financing, and institutional continuity. Recommendations aligned with the PAHO malaria elimination framework are provided.

In malaria-endemic regions, repeated episodes of uncomplicated malaria during infancy are common; however, their associations with neurodevelopment remain unclear. We examined relationships between malaria frequency in infancy and neurodevelopmental outcomes in early and middle childhood, using data from the APEC birth cohort nested within the MiPPAD birth cohort study of Benin. Children were enrolled between January 2010 and June 2011 and followed from birth to 24 months of age. Malaria was assessed at scheduled visits at 6, 9 and 12 months using both PCR and microscopy, and at unscheduled visits for symptomatic episodes using only microscopy; haemoglobin was measured concurrently. Neurodevelopment was assessed at age 1 and 6 years. Analyses included 311 children at 1-year and 248 at 6-year assessments. Malaria exposure was defined as the number of episodes from birth to 1-year neurodevelopmental assessment for three measures: (i) total malaria (PCR- or microscopy-positive), (ii) symptomatic malaria (positive with fever) and (iii) malaria with anaemia (positive with haemoglobin < 11.0 g/dL). Associations with neurodevelopment and haemoglobin's mediating role were assessed using adjusted generalized linear models and mediation analysis. We found that a higher number of symptomatic malaria episodes during infancy was associated with lower motor scores at age one (β: -2.81, [-4.73, -0.89]) and age six (β: -1.43, [-2.65, -0.22]), partially and potentially mediated by haemoglobin concentration. Malaria with anaemia was associated with lower motor scores at age one (β: -1.57, [-3.04, -0.10]) and age six (β: -0.96, [-1.86, -0.05]). No consistent associations were observed with cognitive outcomes. These findings indicate that uncomplicated malaria in infancy is an important risk factor for impaired child development and should not be overlooked. Integrating anaemia screening and management into malaria control and routine child health services may help mitigate long-term developmental impacts in malaria-endemic settings.

IntroductionUncomplicated malaria (UM) remains a major public health concern in Cameroon. This study assessed knowledge, attitudes, prevention practices and care-seeking behaviours related to UM in Kumba and Muyuka, Southwest Region.MethodsA cross-sectional survey was conducted between March and June 2025 among adults (≥18 years), residing in the area for at least 3 months. Multistage sampling yielded 638 valid responses in Kumba and 606 in Muyuka. Data were collected using structured interviewer-administered questionnaires. Descriptive statistics, Pearson correlation (r), Pearson χ2 tests, analysis of variance and multivariate logistic regression were used to evaluate the Knowledge-Attitude-Practice (KAP) pathway and identify predictors of good and appropriate preventive and care-seeking practices.ResultsComprehensive knowledge was poor: only 13.0% in Kumba and 14.9% in Muyuka demonstrated good knowledge although fever was widely recognised (86.1%). Long-lasting insecticidal net ownership was significantly higher in Kumba (81.7%) than in Muyuka (57.1%). Correlation analysis revealed that while knowledge and attitudes were independently associated with practices, knowledge did not significantly correlate with attitudes (r=0.045, p=0.114). In multivariate analysis, younger age, male gender, good knowledge, positive attitudes and good access to malaria information emerged as significant independent predictors of good preventive practices, with specific drivers varying between municipalities.ConclusionDespite high recognition of fever as a symptom, comprehensive malaria knowledge remains low. The weak association between knowledge and attitudes suggests that information alone does not drive behaviour change. Malaria control requires integrated, culturally sensitive strategies that address sociodemographic variations and contextual barriers rather than relying solely on information campaigns.Clinical trial registrationNot applicable.

Prompt diagnosis and treatment of malaria remain central to malaria control strategies. The World Health Organization recommends testing suspected malaria cases before treatment using rapid diagnostic tests (RDTs) or microscopy. However, evidence on the readiness of health facilities to implement this policy in high-burden Nigerian states remains limited. This study assessed facility readiness, testing-before-treatment (TBT) coverage, and facility-level determinants of malaria diagnostic readiness among public health facilities in Kaduna and Kano States, Nigeria. A cross-sectional health facility assessment was conducted across 418 public health facilities in Kaduna and Kano States between August 5 and 28, 2025. A stratified two-stage sampling approach was used, with primary health facilities randomly selected proportionally across all 67 Local Government Areas and all secondary facilities purposively included. Data were collected using a structured Health Facility Assessment questionnaire. Facility readiness was assessed using five indicators: availability of RDTs, availability of microscopy services, trained staff on malaria case management, availability of standard operating procedures, and absence of stock-outs of RDTs or artemisinin-based combination therapy within the previous three months. A composite readiness index (0-5) was constructed and facilities were classified as having full or partial readiness. Associations between facility characteristics and readiness were examined using multivariable logistic regression. All facilities had RDTs and trained staff on malaria case management, and none reported commodity stock-outs during the three-month reference period. Overall, 381 facilities (91.1%) demonstrated full readiness, with a mean readiness score of 4.91 ± 0.29. Microscopy services were available in 23.0% of facilities. Most facilities reported high TBT coverage, with 90.4% reporting ≥ 85% testing coverage. Secondary facilities (aOR = 2.15, 95% CI:1.06-4.36), urban location (aOR = 1.78, 95% CI:1.01-3.12), supportive supervision (aOR = 2.54, 95% CI:1.28-5.02), malaria focal persons (aOR = 2.92, 95% CI:1.63-5.23), and availability of free malaria commodities (aOR = 1.98, 95% CI:1.07-3.68) were significantly associated with full readiness. Public health facilities assessed in Kaduna and Kano States demonstrated high structural readiness for malaria diagnosis. Study limitations include the cross-sectional design, reliance on facility records which may be subject to reporting bias, and the exclusion of private health facilities, limiting generalizability. These findings highlight the need for sustained commodity security, regular supportive supervision, and targeted investments in rural facilities to maintain and strengthen diagnostic readiness.

The creation of transgenic Plasmodium falciparum lines with robust fluorescence across the entire life cycle is essential for advancing our understanding of parasite biology, which in turn informs the development of new drugs and vaccines. In this study, we utilized Plasmodium-optimized genome editing to integrate an mCherry expression cassette into a selected intergenic locus without gene disruption. The resulting marker-free line, NF54-mCh, exhibited intense fluorescence throughout all developmental stages, including asexual and sexual blood stages, as well as mosquito (ookinete, oocyst, and sporozoite) and liver stages. NF54-mCh showed normal proliferation, gametocytogenesis, and efficient transmission to mosquitoes. The ultra-high brightness in salivary gland sporozoites allowed for the non-invasive identification of infected mosquitoes. Sporozoites remained highly infectious to humanized mouse livers, thus enabling the completion of the full life cycle. NF54-mCh serves as a parental line for performing additional genetic modifications, because the CRISPR/Cas9-based genome editing method is free of introduced drug resistance markers. The broader applicability of this strategy was validated by generating similar reporter lines in Plasmodium species utilized in rodent malaria models. In summary, NF54-mCh represents a unique, versatile platform that will accelerate fundamental research and support the future development of malaria control strategies, including new vaccines and drugs.

Mosquito larval source management remains a critical strategy for malaria control, but traditional methods for mapping breeding sites are labor-intensive and slow. While drones offer a promising tool, their application is often hindered by reliance on costly orthomosaic generation (stitching drone images into precise, georeferenced maps) and multispectral sensors, creating significant barriers for resource-limited health programs. This study developed an alternative workflow using a deep learning approach for water body segmentation. We utilized the DeepLabV3 + architecture with an EfficientNetV2 backbone on still RGB and Grayscale drone imagery, bypassing the need for orthomosaics by directly georeferencing individual images using GPS metadata. The model was trained and evaluated on a dataset of over 4,400 images from Pangandaran Regency, Indonesia, with performance assessed via the Intersection over Union (IoU) metric. Predictions were validated through field inspections to confirm water presence and larval abundance. The model achieved a mean IoU of 0.86 on RGB images and 0.80 on Grayscale data, demonstrating robust performance even with minimal spectral information. Field validation of 47 predicted sites confirmed water presence in all cases (100% confirmation of water presence), with 31.9% found to contain mosquito larvae, including the primary local vectors Anopheles vagus and An. sundaicus. The entire workflow, from cloud-based processing to field localization, was executed without specialized hardware or software. Our findings demonstrate that a deep learning-based analysis of still drone imagery provides a scalable, cost-effective, and rapid alternative to multispectral and orthomosaic-dependent methods for larval habitat mapping. This approach democratizes advanced surveillance technology, significantly shortens the delay between data collection and intervention, and holds great promise for enhancing the efficiency of vector control programs in malaria-endemic regions.