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Background: Rwanda has made significant strides in malaria control. This study reviews malaria epidemiology and control strategies in Rwanda from 2018 to 2023, documenting their impact, persistent gaps and emerging challenges. Methods: Data on Rwanda's malaria context from 2018 to 2023 were obtained through a literature review of peer-reviewed articles and grey literature, including annual reports from the malaria programmes, partners, the African Union, and the World Health Organization (WHO). Specific keywords used for the search included "malaria", "Rwanda", "case management", "control", "treatment", and "prevention". Moreover, epidemiological data for this period was extracted from the Health Management Information System (HMIS). Data analysis was done using R & R-Studio, ANOVA to assess the statistical significance (P < 0.05) of observed trends and T-test to compare the focal and blanket IRS techniques. Results/Discussion: Between 2018 and 2023, all malaria indicators showed improvement. Malaria incidence dropped from 345 to 40 cases per 1000 persons (P = 0.00292), the severe malaria rate decreased from 112 to 10/100,000 persons (P = 0.018), and the mortality rate fell from 2.72 to 0.258 deaths /100,000 persons (P = 0.00617). Among children under 5 years of age, incidence decreased significantly from 331 to 52/1,000 persons (P = 0.00123), the severe malaria rate dropped from 214 to 29/100,000 persons (P = 0.00399), and mortality declined from 5 to 0.453/100,000 persons (P = 0.00504). Over the same period, key malaria interventions expanded. The proportion of cases treated by CHWs increased significantly, improving access to early diagnosis and treatment (from 13 to 59%), and the new generations of ITNs (PBO and dual-active ingredient nets) were deployed in 9 districts. Since 2019, a blanket spraying technique has been adopted in 12 IRS districts replacing the focal spraying technique contributing to the significant decrease of malaria incidence from 2019 to 2023 (P = 0.0025). However, new challenges have emerged, including the rise of the K13 R561H mutation associated with artemisinin resistance, the spread of insecticide resistance, and limited intervention coverage due to resource constraints. Conclusion: To sustain the progress achieved, it is essential to intensify malaria control efforts, foster compliance with intervention strategies, enhance surveillance systems for timely and effective responses, and secure long-term funding to sustain these measures. [ABSTRACT FROM AUTHOR]

Introduction: Malaria molecular surveillance (MMS) can provide insights into transmission dynamics, guiding national control programs. We previously designed AmpliSeq assays for MMS, which include different traits of interest (resistance markers and pfhrp2/3 deletions), and SNP barcodes to provide population genetics estimates of Plasmodium vivax and Plasmodium falciparum parasites in the Peruvian Amazon. The present study compares the genetic resolution of the barcodes in the AmpliSeq assays with widely used microsatellite (MS) panels to investigate population genetics of Amazonian malaria parasites. Methods: We analyzed 51 P. vivax and 80 P. falciparum samples from three distinct areas in the Loreto region of the Peruvian Amazon: Nueva Jerusalén (NJ), Mazan (MZ), and Santa Emilia (SE). Population genetics estimates and costs were compared using the SNP barcodes (P. vivax : 40 SNPs and P. falciparum: 28 SNPs) and MS panels (P. vivax : 16 MS and P. falciparum : 7 MS). Results: The P. vivax genetic diversity (expected heterozygosity, He) trends were similar for both markers: He MS = 0.68–0.78 (p > 0.05) and He SNP = 0.36–0.38 (p > 0.05). P. vivax pairwise genetic differentiation (fixation index, FST) was also comparable: FST-MS = 0.04–0.14 and FST-SNP = 0.03–0.12 (pairwise p > 0.05). In addition, P. falciparum genetic diversity trends (He MS = 0–0.48, p < 0.05; He SNP = 0–0.09, p < 0.05) and pairwise FST comparisons (FST-MS = 0.14–0.65, FST-SNP = 0.19–0.61, pairwise p > 0.05) were concordant between both panels. For P. vivax , no geographic clustering was observed with any panel, whereas for P. falciparum , similar population structure clustering was observed with both markers, assigning most parasites from NJ to a distinct subpopulation from MZ and SE. We found significant differences in detecting polyclonal infections: for P. vivax , MS identified a higher proportion of polyclonal infections than SNP (69% vs. 33%, p = 3.3 × 10−5), while for P. falciparum , SNP and MS detected similar rates (46% vs. 31%, p = 0.21). The AmpliSeq assay had a higher estimated per-sample cost compared to MS ($183 vs. $27–49). Discussion: The SNP barcodes in the AmpliSeq assays offered comparable results to MS for investigating population genetics in P. vivax and P. falciparum populations, despite some discrepancies in determining polyclonality. Given both panels have their respective advantages and limitations, the choice between both should be guided by research objectives, costs, and resource availability. [ABSTRACT FROM AUTHOR]

Introduction: Vietnam's goal to eliminate malaria by 2030 is challenged by the further spread of drug-resistant Plasmodium falciparum malaria to key antimalarials, particularly dihydroartemisinin-piperaquine (DHA-PPQ). Methods: The custom targeted NGS amplicon sequencing assay, AmpliSeq Pf Vietnam v2, targeting drug resistance, population genetic- and other markers, was applied to detect genetic diversity and resistance profiles in samples from 8 provinces in Vietnam (n = 354), in a period of steep decline of incidence (2018–2020). Variants in 14 putative resistance genes, including P. falciparum Kelch 13 (PfK13) and P. falciparum chloroquine resistance transporter (Pfcrt) , were analyzed and within-country parasite diversity was evaluated. Other targets included KEL1-lineage markers and diagnostic markers of Pfhrp2/3. Results: A concerning level of DHA-PPQ resistance was detected. The C580Y mutation in PfK13 was found in nearly 80% of recent samples, a significant rise from previous data. Vietnam has experienced a significant challenge with the spread of DHA-PPQ resistant malaria parasites, particularly in the provinces of Binh Phuoc and Gia Lai. Resistance spread to high levels in Binh Thuan prior to the country-wide treatment policy change from DHA-PPQ to pyronadine-artesunate (PA). A complex picture of PPQ-resistance dynamics was observed, with an increase of PPQ-resistance associated Pfcrt mutations, indicating an evolutionary response to antimalarial pressure. Additionally, the compensatory mutation C258W in Pfcrt , which increases chloroquine (CQ) resistance while reversing PPQ resistance, is emerging in Gia Lai following the adoption of PA as the first-line treatment. This study found high levels of multidrug resistance, with over 70% of parasites in 6 out of 8 provinces showing significant sulfadoxine-pyrimethamine (SP) resistance and widespread chloroquine-resistant Pfcrt haplotypes. We also report an absence of P. falciparum histidine rich protein 2 and 3 (Pfhrp2/3) gene deletions, ensuring the continued reliability of HRP2/3-based rapid diagnostic tests. P. falciparum populations in Vietnam are becoming more isolated, with clonal populations showing high geographical clustering by province. The central highlands, particularly Gia Lai province, have the highest residual malaria burden but exhibit low diversity and clonal populations, likely due to the pressures from the antimalarial drugs and targeted national malaria control program (NMCP) efforts. Discussion: In conclusion, examining a broad panel of full-length resistance genes and SNPs provided high-resolution insights into genetic diversity and resistance evolution in Vietnam, offering valuable information to inform local treatment and intervention strategies. [ABSTRACT FROM AUTHOR]

Background: Plasmodium vivax is the most predominant malaria species in Latin America, constituting 71.5% of malaria cases in 2021. With several countries aiming for malaria elimination, it is crucial to prioritize effectiveness of national control programs by optimizing the utilization of available resources and strategically implementing necessary changes. To support this, there is a need for innovative approaches such as genomic surveillance tools that can investigate changes in transmission intensity, imported cases and sources of reintroduction, and can detect molecular markers associated with drug resistance. Methodology/Principal findings: Here, we apply a modified highly-multiplexed deep sequencing assay: Pv AmpliSeq v2 Peru. The tool targets a newly developed 41-SNP Peru barcode for parasite population analysis within Peru, the 33-SNP vivaxGEN-geo panel for country-level classification, and 11 putative drug resistance genes. It was applied to 230 samples from the Peruvian Amazon (2007–2020), generating baseline surveillance data. We observed a heterogenous P. vivax population with high diversity and gene flow in peri-urban areas of Maynas province (Loreto region) with a temporal drift using all SNPs detected by the assay (nSNP = 2909). In comparison, in an indigenous isolated area, the parasite population was genetically differentiated (FST = 0.07–0.09) with moderate diversity and high relatedness between isolates in the community. In a remote border community, a clonal P. vivax cluster was identified, with distinct haplotypes in drug resistant genes and ama1, more similar to Brazilian isolates, likely representing an introduction of P. vivax from Brazil at that time. To test its applicability for Latin America, we evaluated the SNP Peru barcode in P. vivax genomes from the region and demonstrated the capacity to capture local population clustering at within-country level. Conclusions/Significance: Together this data shows that P. vivax transmission is heterogeneous in different settings within the Peruvian Amazon. Genetic analysis is a key component for regional malaria control, offering valuable insights that should be incorporated into routine surveillance. Author summary: Latin America is aiming towards malaria elimination. Genomic surveillance is crucial for a country's malaria strategy, aiding in understanding and stopping the spread of the disease. While widely used for another malaria species (Plasmodium falciparum), limited tools exist for tracking P. vivax, a significant player in malaria-endemic areas outside of Africa, and the primary cause of malaria in Latin America. In this study, we used a new tool, Pv AmpliSeq v2 Peru assay, to examine the genetic makeup of malaria parasites in the Peruvian Amazon. This tool helps us see how the parasites from different areas are connected and tracks markers that could indicate resistance to drugs. We found that the parasites from remote areas in the Amazon were genetically different from parasites in areas surrounding the main city of Iquitos, and parasites in a remote border community were genetically more similar to Brazilian parasites. We also show that the Pv AmpliSeq v2 Peru assay can be used to study parasites from other countries in Latin America, highlighting the broader application in the region. Considering that parasites are not constrained by borders and can easily spread between neighboring countries, a regional approach can be crucial for malaria elimination. [ABSTRACT FROM AUTHOR]

Pathogen genomic epidemiology has the potential to provide a deep understanding of population dynamics, facilitating strategic planning of interventions, monitoring their impact, and enabling timely responses, and thereby supporting control and elimination efforts of parasitic tropical diseases. Plasmodium vivax, responsible for most malaria cases outside Africa, shows high genetic diversity at the population level, driven by factors like sub‐patent infections, a hidden reservoir of hypnozoites, and early transmission to mosquitoes. While Latin America has made significant progress in controlling Plasmodium falciparum, it faces challenges with residual P. vivax. To characterize genetic diversity and population structure and dynamics, we have analyzed the largest collection of P. vivax genomes to date, including 1474 high‐quality genomes from 31 countries across Asia, Africa, Oceania, and America. While P. vivax shows high genetic diversity globally, Latin American isolates form a distinctive population, which is further divided into sub‐populations and occasional clonal pockets. Genetic diversity within the continent was associated with the intensity of transmission. Population differentiation exists between Central America and the North Coast of South America, vs. the Amazon Basin, with significant gene flow within the Amazon Basin, but limited connectivity between the Northwest Coast and the Amazon Basin. Shared genomic regions in these parasite populations indicate adaptive evolution, particularly in genes related to DNA replication, RNA processing, invasion, and motility – crucial for the parasite's survival in diverse environments. Understanding these population‐level adaptations is crucial for effective control efforts, offering insights into potential mechanisms behind drug resistance, immune evasion, and transmission dynamics. [ABSTRACT FROM AUTHOR]

Although the power of genetic surveillance tools has been acknowledged widely, there is an urgent need in malaria endemic countries for feasible and cost-effective tools to implement in national malaria control programs (NMCPs) that can generate evidence to guide malaria control and elimination strategies, especially in the case of Plasmodium vivax. Several genetic surveillance applications (‘use cases’) have been identified to align research, technology development, and public health efforts, requiring different types of molecular markers. Here we present a new highly-multiplexed deep sequencing assay (Pv AmpliSeq). The assay targets the 33-SNP vivaxGEN-geo panel for country-level classification, and a newly designed 42-SNP withincountry barcode for analysis of parasite dynamics in Vietnam and 11 putative drug resistance genes in a highly multiplexed NGS protocol with easy workflow, applicable for many different genetic surveillance use cases. The Pv AmpliSeq assay was validated using: 1) isolates from travelers and migrants in Belgium, and 2) routine collections of the national malaria control program at sentinel sites in Vietnam. The assay targets 229 amplicons and achieved a high depth of coverage (mean 595.7 ± 481) and high accuracy (mean error-rate of 0.013 ± 0.007). P. vivax parasites could be characterized from dried blood spots with a minimum of 5 parasites/μL and 10% of minority-clones. The assay achieved good spatial specificity for between-country prediction of origin using the 33- SNP vivaxGEN-geo panel that targets rare alleles specific for certain countries and regions. A high resolution for within-country diversity in Vietnam was achieved using the designed 42-SNP within-country barcode that targets common alleles (median MAF 0.34, range 0.01-0.49. Many variants were detected in (putative) drug resistance genes, with different predominant haplotypes in the pvmdr1 and pvcrt genes in different provinces in Vietnam. The capacity of the assay for high resolution identity-by-descent (IBD) analysis was demonstrated and identified a high rate of shared ancestry within Gia Lai Province in the Central Highlands of Vietnam, as well as between the coastal province of Binh Thuan and Lam Dong. Our approach performed well in geographically differentiating isolates at multiple spatial scales, detecting variants in putative resistance genes, and can be easily adjusted to suit the needs in other settings in a country or region. We prioritize making this tool available to researchers and NMCPs in endemic countries to increase ownership and ensure data usage for decision-making and malaria policy. [ABSTRACT FROM AUTHOR]

Monitoring the genetic structure of pathogen populations may be an economical and sensitive approach to quantify the impact of control on transmission dynamics, highlighting the need for a better understanding of changes in population genetic parameters as transmission declines. Here we describe the first population genetic analysis of two major human malaria parasites, Plasmodium falciparum (Pf) and Plasmodium vivax (Pv), following nationwide distribution of long‐lasting insecticide‐treated nets (LLINs) in Papua New Guinea (PNG). Parasite isolates from pre‐ (2005–2006) and post‐LLIN (2010–2014) were genotyped using microsatellite markers. Despite parasite prevalence declining substantially (East Sepik Province: Pf = 54.9%–8.5%, Pv = 35.7%–5.6%, Madang Province: Pf = 38.0%–9.0%, Pv: 31.8%–19.7%), genetically diverse and intermixing parasite populations remained. Pf diversity declined modestly post‐LLIN relative to pre‐LLIN (East Sepik: Rs = 7.1–6.4, HE = 0.77–0.71; Madang: Rs = 8.2–6.1, HE = 0.79–0.71). Unexpectedly, population structure present in pre‐LLIN populations was lost post‐LLIN, suggesting that more frequent human movement between provinces may have contributed to higher gene flow. Pv prevalence initially declined but increased again in one province, yet diversity remained high throughout the study period (East Sepik: Rs = 11.4–9.3, HE = 0.83–0.80; Madang: Rs = 12.2–14.5, HE = 0.85–0.88). Although genetic differentiation values increased between provinces over time, no significant population structure was observed at any time point. For both species, a decline in multiple infections and increasing clonal transmission and significant multilocus linkage disequilibrium post‐LLIN were positive indicators of impact on the parasite population using microsatellite markers. These parameters may be useful adjuncts to traditional epidemiological tools in the early stages of transmission reduction. [ABSTRACT FROM AUTHOR]

Background: In Vietnam, malaria transmission has been reduced to very low levels over the past 20 years, and as a consequence, the country aims to eliminate malaria by 2030. This study aimed to characterize the dynamics and extent of the parasite reservoir in Central Vietnam, in order to further target elimination strategies and surveillance. Methods: A 1-year prospective cohort study (n = 429) was performed in three rural communities in Quang Nam province. Six malaria screenings were conducted between November 2014 and November 2015, including systematic clinical examination and blood sampling for malaria parasite identification, as well as molecular and serological analysis of the study population. Malaria infections were detected by light microscopy (LM) and quantitative real time PCR (qPCR), while exposure to Plasmodium falciparum and Plasmodium vivax was measured in the first and last survey by ELISA for PfAMA1, PfGLURP R2, PvAMA1, and PvMSP1-19. Classification and regression trees were used to define seropositivity and recent exposure. Results: Four malaria infections (2 P. falciparum, 2 P. vivax) were detected in the same village by qPCR and/or LM. No fever cases were attributable to malaria. At the same time, the commune health centre (serving a larger area) reported few cases of confirmed malaria cases. Nevertheless, serological data proved that 13.5% of the surveyed population was exposed to P. falciparum and/or P. vivax parasites during the study period, of which 32.6% were seronegative at the start of the study, indicating ongoing transmission in the area. Risk factor analysis for seroprevalence and exposure to P. falciparum and/or P. vivax identified structural or economic risk factors and activity/behaviour-related factors, as well as spatial heterogeneity at the village level. Conclusions: Previous studies in Central Vietnam demonstrated high occurrence of asymptomatic and sub-microscopic infections. However, in this study very few asymptomatic infections were detected despite serological evidence of continued transmission. Nonetheless, the factors associated with spatial heterogeneity in transmission could be evaluated using serological classification of recent exposure, which supports the usefulness of serological methods to monitor malaria transmission. [ABSTRACT FROM AUTHOR]

Introduction: Malaria molecular surveillance (MMS) can provide insights into transmission dynamics, guiding national control programs. We previously designed AmpliSeq assays for MMS, which include different traits of interest (resistance markers and pfhrp2/3 deletions), and SNP barcodes to provide population genetics estimates of Plasmodium vivax and Plasmodium falciparum parasites in the Peruvian Amazon. The present study compares the genetic resolution of the barcodes in the AmpliSeq assays with widely used microsatellite (MS) panels to investigate population genetics of Amazonian malaria parasites., Methods: We analyzed 51  P. vivax and 80  P. falciparum samples from three distinct areas in the Loreto region of the Peruvian Amazon: Nueva Jerusalén (NJ), Mazan (MZ), and Santa Emilia (SE). Population genetics estimates and costs were compared using the SNP barcodes ( P. vivax : 40 SNPs and P. falciparum: 28 SNPs) and MS panels ( P. vivax : 16 MS and P. falciparum : 7 MS)., Results: The P. vivax genetic diversity (expected heterozygosity, He ) trends were similar for both markers: He MS = 0.68-0.78 ( p > 0.05) and He SNP = 0.36-0.38 ( p > 0.05). P. vivax pairwise genetic differentiation (fixation index, F ST ) was also comparable: F ST-MS = 0.04-0.14 and F ST-SNP = 0.03-0.12 (pairwise p > 0.05). In addition, P. falciparum genetic diversity trends ( He MS = 0-0.48, p < 0.05; He SNP = 0-0.09, p < 0.05) and pairwise F ST comparisons (F ST-MS = 0.14-0.65, F ST-SNP = 0.19-0.61, pairwise p > 0.05) were concordant between both panels. For P. vivax , no geographic clustering was observed with any panel, whereas for P. falciparum , similar population structure clustering was observed with both markers, assigning most parasites from NJ to a distinct subpopulation from MZ and SE. We found significant differences in detecting polyclonal infections: for P. vivax , MS identified a higher proportion of polyclonal infections than SNP (69% vs. 33%, p = 3.3 × 10 -5 ), while for P. falciparum , SNP and MS detected similar rates (46% vs. 31%, p = 0.21). The AmpliSeq assay had a higher estimated per-sample cost compared to MS ($183 vs. $27-49)., Discussion: The SNP barcodes in the AmpliSeq assays offered comparable results to MS for investigating population genetics in P. vivax and P. falciparum populations, despite some discrepancies in determining polyclonality. Given both panels have their respective advantages and limitations, the choice between both should be guided by research objectives, costs, and resource availability., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Cabrera-Sosa, Safarpour, Kattenberg, Ramirez, Vinetz, Rosanas-Urgell, Gamboa and Delgado-Ratto.)

Malaria molecular surveillance has great potential to support national malaria control programs (NMCPs), informing policy for its control and elimination. Here, we present a new three-day workflow for targeted resequencing of markers in 13 resistance-associated genes, histidine rich protein 2 and 3 (hrp2&3) , a country (Peru)-specific 28 SNP-barcode for population genetic analysis, and apical membrane antigen 1 ( ama1 ), using Illumina short-read sequencing technology. The assay applies a multiplex PCR approach to amplify all genomic regions of interest in a rapid and easily standardizable procedure and allows simultaneous amplification of a high number of targets at once, therefore having great potential for implementation into routine surveillance practice by NMCPs. The assay can be performed on routinely collected filter paper blood spots and can be easily adapted to different regions to investigate either regional trends or in-country epidemiological changes., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (Copyright © 2023 The Authors; This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).)

Molecular surveillance for malaria has great potential to support national malaria control programs (NMCPs). To bridge the gap between research and implementation, several applications (use cases) have been identified to align research, technology development, and public health efforts. For implementation at NMCPs, there is an urgent need for feasible and cost-effective tools. We designed a new highly multiplexed deep sequencing assay (Pf AmpliSeq), which is compatible with benchtop sequencers, that allows high-accuracy sequencing with higher coverage and lower cost than whole-genome sequencing (WGS), targeting genomic regions of interest. The novelty of the assay is its high number of targets multiplexed into one easy workflow, combining population genetic markers with 13 nearly full-length resistance genes, which is applicable for many different use cases. We provide the first proof of principle for hrp2 and hrp3 deletion detection using amplicon sequencing. Initial sequence data processing can be performed automatically, and subsequent variant analysis requires minimal bioinformatic skills using any tabulated data analysis program. The assay was validated using a retrospective sample collection ( n  = 254) from the Peruvian Amazon between 2003 and 2018. By combining phenotypic markers and a within-country 28-single-nucleotide-polymorphism (SNP) barcode, we were able to distinguish different lineages with multiple resistance haplotypes (in dhfr, dhps, crt and mdr1 ) and hrp2 and hrp3 deletions, which have been increasing in recent years. We found no evidence to suggest the emergence of artemisinin (ART) resistance in Peru. These findings indicate a parasite population that is under drug pressure but is susceptible to current antimalarials and demonstrate the added value of a highly multiplexed molecular tool to inform malaria strategies and surveillance systems. IMPORTANCE While the power of next-generation sequencing technologies to inform and guide malaria control programs has become broadly recognized, the integration of genomic data for operational incorporation into malaria surveillance remains a challenge in most countries where malaria is endemic. The main obstacles include limited infrastructure, limited access to high-throughput sequencing facilities, and the need for local capacity to run an in-country analysis of genomes at a large-enough scale to be informative for surveillance. In addition, there is a lack of standardized laboratory protocols and automated analysis pipelines to generate reproducible and timely results useful for relevant stakeholders. With our standardized laboratory and bioinformatic workflow, malaria genetic surveillance data can be readily generated by surveillance researchers and malaria control programs in countries of endemicity, increasing ownership and ensuring timely results for informed decision- and policy-making.

The Plasmodium vivax reticulocyte invasion process is still poorly understood, with only a few receptor-ligand interactions identified to date. Individuals with the Southeast Asian ovalocytosis (SAO) phenotype have a deletion in the band 3 protein on the surface of erythrocytes, and are reported to have a lower incidence of clinical P. vivax malaria. Based on this observation, band 3 has been put forward as a receptor for P. vivax invasion, although direct proof is still lacking. In this study, we combined functional ex vivo invasion assays and transcriptome sequencing to uncover a band 3-mediated invasion pathway in P. vivax and potential band 3 ligands. Invasion by P. vivax field isolates was 67%-71% lower in SAO reticulocytes compared with non-SAO reticulocytes. Reticulocyte invasion was decreased by 40% and 27%-31% when blocking with an anti-band 3 polyclonal antibody and a PvTRAg38 peptide, respectively. To identify new band 3 receptor candidates, we mRNA-sequenced schizont-stage isolates used in the invasion assays, and observed high transcriptional variability in multigene and invasion-related families. Transcriptomes of isolates with low or high dependency on band 3 for invasion were compared by differential expression analysis, which produced a list of band 3 ligand candidates with high representation of PvTRAg genes. Our ex vivo invasion assays have demonstrated that band 3 is a P. vivax invasion receptor and confirm previous in vitro studies showing binding between PvTRAg38 and band 3, although the lower and variable inhibition levels observed suggest the involvement of other ligands. By coupling transcriptomes and invasion phenotypes from the same isolates, we identified a list of band 3 ligand candidates, of which the overrepresented PvTRAg genes are the most promising for future research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 De Meulenaere, Prajapati, Villasis, Cuypers, Kattenberg, Kasian, Laman, Robinson, Gamboa, Laukens and Rosanas-Urgell.)

We report an outbreak investigation of two fatal cases of autochthonous Plasmodium falciparum malaria that occurred in Belgium in September 2020. Various hypotheses of the potential source of infection were investigated. The most likely route of transmission was through an infectious exotic Anopheles mosquito that was imported via the international airport of Brussels or the military airport Melsbroek and infected the cases who lived at 5 km from the airports. Based on genomic analysis of the parasites collected from the two cases, the most likely origin of the Plasmodium was Gabon or Cameroon. Further, the parasites collected from the two Belgian patients were identical by descent, which supports the assumption that the two infections originated from the bite of the same mosquito, during interrupted feeding. Although airport malaria remains a rare event, it has significant implications, particularly for the patient, as delayed or missed diagnosis of the cause of illness often results in complications and mortality. Therefore, to prevent such severe or fatal outcomes, we suggest a number of public health actions including increased awareness among health practitioners, especially those working in the vicinity of airports, and increased surveillance of exotic mosquito species at airports.