After substantial progress in malaria control, Ethiopia's Amhara Region experienced a marked resurgence since 2018. The relative contributions of climate variability, environmental context, intervention coverage, and unmeasured factors to this resurgence remain inadequately quantified. This study used a Bayesian spatiotemporal framework to estimate factor associations with malaria incidence, decompose spatial versus temporal climate effects, and identify persistent hotspots. We conducted an ecological district-level panel analysis of 13,944 district-month observations from 166 districts (January 2018-December 2024). Monthly confirmed malaria counts (total,Plasmodium falciparum,P. vivax) were modelled using Bayesian hierarchical negative binomial regression with BYM2 spatial and AR(1) temporal random effects, fitted with integrated nested Laplace approximation. Covariates included lagged rainfall, temperature, NDVI, elevation, and programmatic indicators (ITN ownership, IRS protection, and larval source management [LSM] intensity). Climate covariates were decomposed into between-district (spatial) means and within-district (temporal) deviations. Sensitivity analyses included alternative IRS protection windows and district fixed-effects models. A total of 5,746,571 confirmed cases were reported (64.3%P. falciparum, 35.7%P. vivax). Mean monthly incidence increased 5.5-fold from 1.19 per 1,000 (2018) to 6.53 per 1,000 (2024), while regional mean maximum temperature showed a small declining trend over the period. In fully adjusted models, higher lagged maximum temperature and rainfall were associated with higher incidence, and elevation was protective. IRS protection, higher ITN ownership, and higher LSM intensity were each associated with lower incidence; effect directions were consistent in within-district sensitivity analyses, although residual confounding and measurement error cannot be excluded. Climate-incidence associations were predominantly spatial (between-district) rather than temporal (within-district), suggesting that geographic ecological suitability explains much of the spatial patterning, rather than temporal warming trends explaining the resurgence. Districts with persistently elevated residual spatial risk (exceedance probability of residual RR > 1.25) clustered in low-elevation western border areas. Malaria resurgence in Amhara (2018-2024) occurred alongside strong spatial climatic and elevational gradients and was not consistent with a temporal warming-driven explanation at the regional scale. Remaining unexplained spatiotemporal variation highlights the likely importance of unmeasured drivers (e.g., conflict-related service disruption, vector/insecticide resistance dynamics, and population mobility). Climate-informed, spatially targeted intervention packages prioritizing districts with persistently high residual risk are warranted.

Abstract Background India has made substantial progress in reducing Plasmodium falciparum malaria cases and has set a target to eliminate malaria by 2030. Although ACT treatment remains effective, tracking regional differences in genetic variants associated with antimalarial resistance is required for effective drug policy implementation. Methods We analysed 238 P. falciparum clinical samples from six Indian states by sequencing 15 parasite genes associated with reduced drug effectiveness. The method involved nanopore sequencing of target gene amplicons derived from dried blood spots using a highly sensitive PfMDR15 surveillance panel. Results India’s historical policy of artesunate-sulfadoxine-pyrimethamine in central India and artemether-lumefantrine in the Northeast has shaped contrasting resistance profiles. In the Northeast, chloroquine resistance persisted at high frequency (Pfcrt K76T and CVIET haplotype; Pfaat1 S258L), alongside quintuple and sextuple Pfdhfr–Pfdhps haplotypes conferring complete sulfadoxine-pyrimethamine resistance. Central India showed variable chloroquine resistance (parasites largely retained wild-type Pfcrt) and emerging lumefantrine tolerance (Pfmdr1 Y184F, Pfaat1 S258L). Interestingly, Delhi (Central India) parasites resembled profiles from the distant Northeast, which borders South East Asia. The detection of Pfaat1 S258L, previously reported only from Africa and associated with reduced lumefantrine susceptibility, suggests convergent evolution under ACT partner-drug pressure. No WHO-validated Pfk13 artemisinin resistance mutations were detected, supporting continued efficacy of artemisinin-based combination therapy (ACT). Conclusion India’s resistance landscape is fragmented, with signals of expanding lumefantrine tolerance and importation or evolution of globally relevant mutations. These findings highlight the importance of integrating molecular genomic surveillance into malaria control policy to monitor and protect ACT effectiveness and advance malaria elimination.

TEpiNom: A computational framework integrating population data to prioritize plasmodium falciparum T cell epitopes.

Inhibiting catalytic activity of Plasmodium falciparum aspartate protease plasmepsin V: A biochemical approach to malaria intervention.

Cytokine-induced killer (CIK) cells inhibit Plasmodium falciparum parasitemia through cytolytic effector activity in vitro.

Invasion and development of Plasmodium falciparum in erythroblasts of humans carrying G6PD viangchan.

In vitro antiplasmodial activity, acute toxicity and phytochemical quantification of selected medicinal plants used for the management of uncomplicated malaria in eastern Uganda.

Bioprospecting of the Amazonian mapati fruit (Pourouma cecropiifolia Martius): Antiproliferative and antimalarial potential of peel and seed hydroethanolic extracts.

Three new single nucleotide polymorphisms identified in Plasmodium falciparum isolates from Kogi State, Nigeria.

Improved quantitative structure-activity relationship (QSAR) models to predict the activity of hydroxamic acids as HDAC inhibitors against malaria parasites.

Structure-Based Design of New Pyrazole Inhibitors Targeting Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH)

Despite ongoing malaria control efforts, the disease remains a major cause of morbidity and mortality in Nigeria. As transmission declines in many endemic settings, asymptomatic and submicroscopic infections increasingly constitute a hidden reservoir that sustains malaria transmission. These infections often escape detection by routine diagnostic methods yet remain epidemiologically significant, particularly in elimination-focused programmes. This study assessed the prevalence of asymptomatic and submicroscopic malaria parasitaemia among individuals attending selected health facilities in Jos, Plateau State, Nigeria. A cross-sectional survey was conducted among 400 apparently healthy individuals across five selected health facilities in Jos, Plateau State, Nigeria. Participants aged ≥6 months who exhibited no clinical signs or symptoms of malaria were enrolled. Malaria parasitaemia was confirmed by Giemsa-stained microscopy, submicroscopic Plasmodium infection by 18S mRNA using nested PCR. Out of 400 participants screened, 78 (19.5%) tested positive for malaria by microscopy, with prevalence varying across health facilities from 11.2% to 30.0%. Males had a significantly higher infection prevalence (26.0%) compared to females (15.7%). Molecular analysis revealed a substantially higher burden of infection: among 120 microscopy-negative samples analysed by nPCR, 74 (61.7%) were positive, indicating a high prevalence of submicroscopic malaria parasitaemia. Although submicroscopic infections did not differ significantly across demographic groups (p > 0.05), the highest prevalence was observed among pregnant women (64.5%) and children (66.7%). Plasmodium falciparum was the predominant species (55.9%), followed by P. ovale (16.5%), P. malariae (6.5%), and P. vivax (5.3%). Asymptomatic and submicroscopic malaria infections constitute a substantial hidden reservoir in Jos, Nigeria. Reliance solely on routine diagnostic methods may lead to significant underestimation of malaria prevalence. Integrating sensitive molecular diagnostics into surveillance systems is critical for effective malaria elimination in low-to-moderate-transmission settings.

Plasmodium falciparum and P. vivax are parasites responsible for most malaria cases globally. In areas where these species co-exist, individuals gain protection from P. vivax more rapidly, and important biological differences between species may impact the immune response. CD4 T cells are key drivers of immunity to malaria, both as effector and helper cells, with T-follicular helper (Tfh) cells having key roles in antibody development. Comparative studies on CD4 T cell responses between these species are limited. We assessed CD4 T cells in adults with either P. falciparum or P. vivax malaria. Activation and proliferation of CD4 T cells were measured ex vivo, and functional capacity was determined by intracellular cytokine staining using flow cytometry. The phenotype, activation and proliferation of CD4 T cells and effector CD4 T cell subsets were comparable between species. However, within the peripheral (p)Tfh cell compartment, there was some evidence for species-dependent activation with relative increased pTfh1 cells in P. falciparum infection. Additionally, in P. falciparum, increased IL-10 production was detected, including within IL-21 producing CD4 T cells. While activation and function of CD4 T cells in malaria are largely comparable, some species-dependent responses are detected within the pTfh cell compartment that may impact antibody development.

Malaria and COVID-19 co-infections pose a major clinical challenge, as overlapping symptoms can lead to misdiagnosis and delays in treatment. Emerging evidence suggests that SARS-CoV-2 may influence malaria pathogenesis through dysregulation of the renin-angiotensin system. This study assessed clinical, biochemical, and immunological alterations associated with single and dual infections. A total of 96 participants aged 15-64 years were enrolled and classified into four groups: COVID-19 (n = 28), malaria (n = 28), co-infection (n = 16), and healthy controls (n = 24). Blood and nasopharyngeal samples were tested using rapid diagnostic tests, microscopy, and RT-PCR. Disease severity biomarkers were quantified using spectrophotometry and ELISA. Statistical analyses were performed in GraphPad Prism version 9.0, with significance set at p < 0.05. Co-infected participants exhibited significantly elevated biochemical markers (ALT, AST, urea, creatinine, and erythropoietin) compared to all other groups. Co-infection also triggered robust increases in IFN-γ and IL-1β, whereas malaria alone was associated with higher IL-6, IL-4, and IL-10, and COVID-19 alone was associated with elevated IL-2 and TNF-α. ANG2 levels were highest in both COVID-19 and co-infected groups, while ACE2 was markedly elevated in COVID-19 (p < 0.01). Correlation analyses revealed distinct biomarker networks driven by parasitaemia and viral load, implicating pathways linked to inflammation, erythropoiesis, and endothelial dysfunction. Notably, ACE2 demonstrated strong discriminatory power for predicting disease severity, with AUCs of 0.77 for malaria and 0.85 for COVID-19. These findings underscore the diagnostic and prognostic value of vascular and immune biomarkers for early risk stratification, particularly in malaria-COVID-19 co-infection, and may guide improved clinical management in co-endemic regions.

Iron deficiency, anemia, and Plasmodium infection are global health challenges with overlapping geographical distributions, particularly affecting pregnant women in Africa, yet the mechanisms underlying their interactions remain poorly understood. We used a multilayered approach combining clinical data from Malawian pregnant women (n=711) in the REVAMP trial, a genetic mouse model [Tmprss6-knockout (KO)], and in vitro Plasmodium falciparum cultures to clarify iron-malaria associations. Iron deficiency was associated with 50% reduced P. falciparum parasitemia in pregnant women [95% CI (30 to 64%), P<0.0001], while iron-deficient mice exhibited improved survival against P. berghei (median 15.5 days versus 7.0 days for WT mice) and protection from cerebral malaria (83% versus 17% survival). Iron chelation substantially changed the transcriptomic and proteomic profile of cultured P. falciparum parasites. Intravenous iron supplementation did not increase parasitemia when coupled with malaria prevention. These findings demonstrate that iron deficiency protects against Plasmodium infection and support World Health Organization recommendations for iron supplementation in malaria-endemic regions when combined with adequate malaria prevention strategies in place.

Genetic metrics derived from Plasmodium falciparum infections offer a potential complement to conventional malaria surveillance by utilizing features of parasite diversity and relatedness to estimate transmission intensity. However, the performance of genetic metrics to predict epidemiologic metrics across a wide range of transmission settings remains understudied. Dried blood spots from 3563 symptomatic malaria cases were collected from 26 sentinel health facilities across Uganda during two collections in 2023. Amplicon deep sequencing of 165 polyallelic microhaplotypes was performed using MAD4HatTeR. Within-host diversity metrics (complexity of infection (COI), effective complexity of infection (eCOI), percent polyclonality, within-host relatedness) and between-host relatedness metrics were calculated. Associations with prevalence and recent incidence were evaluated using correlation and regression analyses, and estimation accuracy was examined using nested grouped cross-validation. Marked geographic heterogeneity in malaria burden was evident across sites; parasite prevalence ranged from 5.0 to 49.23% in Round 1, while incidence ranged from 91 to 1062 cases per 1000 person-years (PY) in Round 1 and 33 to 1667 cases per 1000 PY in Round 2. COI and eCOI had a strong positive association with parasite prevalence. The proportion of highly related infection pairs was negatively associated with both prevalence and incidence and was the genetic metric most consistently associated with incidence. Nested grouped cross-validation identified single-predictor models using COI or eCOI as optimal for estimating prevalence, yielding a pooled cross-validated correlation of r = 0.79. Models estimating incidence showed weaker performance, with models incorporating both diversity and relatedness metrics achieving a pooled correlation of r = 0.37. Microhaplotype-based metrics of within-host diversity, particularly COI and eCOI, reliably reflected spatial variation in malaria prevalence across Uganda, while between-host relatedness provided complementary information and was the strongest predictor of incidence. These findings indicate that parasite genomic metrics derived from polyallelic microhaplotypes can capture broad differences in transmission intensity reflected by parasite prevalence, but may have more limited ability to predict incidence. Integration of genomic metrics with harmonized epidemiologic data and expanded sampling of asymptomatic infections will be important next steps to understand the potential utility of parasite genetic metrics for malaria surveillance and subnational stratification.

Malaria remains a critical parasitic disease in tropical regions, with environmental temperature significantly influencing the development and transmission of Plasmodium falciparum. While low temperature triggers gametocyte differentiation in mosquito, the molecular mechanisms underlying temperature-responsive chromatin and transcriptional dynamics in asexual stages (ring and trophozoite) remain unclear. This study integrates Assay for Transposase-Accessible Chromatin with Sequencing (ATAC-seq) and RNA-sequencing (RNA-seq) to characterize genome-wide chromatin accessibility and gene expression profiles in P.falciparum under human body temperature (37°C) and mosquito-mimicking temperature (26°C). Synchronized ring (45h post-invasion) and trophozoite (70h post-invasion) stages were subjected to temperature treatments (37°C versus 26°C). ATAC-seq was used to identify accessible chromatin regions, RNA-seq analyzed differentially expressed genes (DEGs), and quantitative real-time polymerase chain reaction (qPCR) validated key gene expression changes. Low temperatures exerts a profound impact on the activation and expression of sexual-stage-specific genes in P.falciparum and induced 1083 differentially accessible regions (DARs) in the ring stage, including 1081 gains and only 2 losses, which were enriched primarily in promoter regions (≤ 3kb upstream of transcription start sites), whereas no significant DARs were detected in the trophozoite stage, indicating stage-specific sensitivity to temperature. Functional analyses revealed DAR-associated genes enriched in host cell membrane interactions, antigenic variation, and pathways such as symbiont-mediated perturbation of host erythrocyte aggregation, with the identification of temperature-responsive transcription factor motifs (e.g., DEAR-3, ERF096). Integration of ATAC-seq and RNA-seq revealed a positive correlation between chromatin accessibility and gene expression, with 41 genes exhibiting concordant DAR-DEG changes, suggesting that dynamic chromatin remodeling regulates temperature-responsive transcription. Low temperature selectively modulates chromatin accessibility and gene expression in the ring stage, while trophozoites show no chromatin reconfiguration, highlighting stage-specific thermal sensitivity. This study represents the first integrative analysis of ATAC-seq and RNA-seq data from P.falciparum under low temperature stress, identifying critical temperature-responsive regulatory regions, providing insights into the parasite's environmental adaptation and a foundational resource of temperature-responsive regulatory regions, whose future functional validation could inform the development of novel, chromatin-targeted antimalarial strategies.

Progression occurs in more than 10% of melanoma patients initially diagnosed with local disease, emphasizing the need for improved risk stratification even in early stages. In this study, we used an enrichment method based on recombinant Plasmodium falciparum VAR2CSA protein to enable the capture of rare circulating tumor cells (CTCs) from a single blood draw in early-stage (AJCC I-II) melanoma patients. CTCs were subsequently identified using fluorescent antibodies targeting tumor initiating and melanoma specific cell markers. In parallel, we investigated circulating tumor DNA (ctDNA) in these patients. Among 92 early-stage melanoma patients, CTCs were detected in 21 patients (22.8%) at time of initial diagnosis. The presence of CTCs was significantly associated with an unfavorable clinical outcome, such as disease progression or death related to melanoma disease, with a median observation time of 30months (IQR 24.0-36.0) (p = 0.043). Furthermore, when combining CTC with ctDNA detection, a highly significant correlation with disease progression was observed (p = 0.014) underlining the role of early tumor cell dissemination in melanoma disease. These findings suggest that CTC assessment, particularly when combined with ctDNA analysis, may represent a valuable biomarker to facilitate risk stratification of early-stage melanoma patients to improve personalized treatment approaches.

Malaria in pregnancy remains a major driver of poor maternal and neonatal health outcomes in sub-Saharan Africa. For decades, intermittent preventive treatment in pregnancy (IPTp), with sulphadoxine-pyrimethamine (SP), has mitigated malaria-associated health risks, but concerns have been raised regarding accumulated Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutations on the efficacy of SP. Western Kenya, including Busia County, is a high malaria transmission setting where molecular surveillance of dhfr and dhps mutations remains limited. This study assessed the prevalence and haplotype structure of dhfr and dhps mutations in P. falciparum isolates from Busia County, Kenya. A total of 66 samples of P. falciparum isolates collected from patients attending Matayos Sub-County Hospital between November 2024 and January 2025 were analysed. PCR amplification and Sanger sequencing targeted dhfr codons C50R, N51I, C59R, S108N/T, I164L, and dhps codons I431V, S436A/F, A437G, K540E, A581G, and A613S/T to determine mutation frequencies, haplotypes, and combined dhps and dhfr haplotype profiles. High frequencies of dhfr and dhps mutations were observed across the parasite isolates. The most common dhfr substitutions included N51I (85.2%) and C59R (75.4%), while S108N (32.8%) and S108T (19.7%) were detected at lower frequencies. Dhfr haplotypes identified included N51I + C59R, N51I + C59R + S108N, and a N51I + C59R + S108T + I164L variant. The I164L mutation was detected at a frequency of 18.0% and was observed exclusively on a non-canonical S108T background (19.7%). Dhps haplotypes were dominated by A437G (92.3%), K540E (40%) alone, and the A437G + K540E double mutant. Combined dhfr and dhps haplotype analyses revealed circulation of classical dhfr triple-mutant (N51I + C59R + S108N) backgrounds with dhps A437G. Quintuple haplotypes (dhfr N51I + C59R + S108T + I164L with dhps A437G) and rare complex haplotypes incorporating both I164L and K540E or I164L and S436F were also detected. These findings indicate the persistence and circulation of both canonical and non-canonical dhfr and dhps haplotypes in P. falciparum isolates from Busia County. This study highlights the need for continuous molecular and phenotypic surveillance to clarify the functional and epidemiological significance of parasites carrying S108T and I164L mutations, and to inform IPT policy.

Understanding the molecular basis of enzyme inhibition is crucial for rational drug design, particularly against parasitic targets such as Plasmodium falciparum plasmepsin II (PlmII), an aspartic protease essential for hemoglobin degradation. In this study, we repurposed fluoroquinolone drugs, namely, ofloxacin, levofloxacin, and moxifloxacin, to inhibit the catalytic activity of mature PlmII (mPlmII). Thermodynamic analyses revealed favorable enthalpic and entropic contributions that correlate with the binding strength of each drug to mPlmII. Detailed enzyme kinetics assays, combined with molecular docking studies, demonstrated that moxifloxacin, with an IC50 value of 0.15 ± 0.02 μM, exhibits the most potent inhibition, primarily through hydrogen bonding with the catalytic dyad, Asp34 and Asp214. Quantum mechanics/molecular mechanics (QM/MM) (ONIOM) calculations using B3LYP/6-31G*: UFF further corroborated this binding mode, with donor-acceptor distances ranging from 2.8 to 3.3 Å, consistent with moderate to strong hydrogen bonding. Notably, methylation of the NH group disrupts these critical interactions, altering the ligand's positioning within the active site and resulting in weakened hydrogen bonds and reduced inhibitory efficacy. Overall, our findings reveal that precise hydrogen bonding with Asp34 and Asp214 is essential for the effective inhibition of mPlmII activity, and even minimal structural modifications, such as NH methylation, can impair active site engagement.