Seasonal Malaria Chemoprevention Therapy in Children Up To 9 Years of Age: Protocol for a Cluster-Randomized Trial Study.

BACKGROUND: Seasonal malaria chemoprevention (SMC) is recommended by the World Health Organization for the sub-Sahel region in sub-Saharan Africa for preventing malaria in children 3 months old to younger than 5 years. Since 2016, the Malian National Malaria Control Program has deployed SMC countrywide during its high malaria transmission season at a rate of 4 monthly cycles annually. The standard SMC regimen includes sulfadoxine-pyrimethamine (SP) plus amodiaquine (AQ). Resistance against SP is suspected to be rising across West Africa; therefore, assessing the effectiveness of an alternative antimalarial drug for SMC is needed to provide a second-line regimen when it is ultimately needed. It is not well understood whether SMC effectively prevents malaria in children aged 5 years or older. OBJECTIVE: The primary goal of the study is to compare 2 SMC regimens (SP-AQ and dihydroartemisinin-piperaquine [DHA-PQ]) in preventing uncomplicated Plasmodium falciparum malaria in children 3 months to 9 years old. Secondly, we will assess the possible use of DHA-PQ as an alternative SMC drug in areas where resistance to SP or AQ may increase following intensive use. METHODS: The study design is a 3-arm cluster-randomized design comparing the SP-AQ and DHA-PQ arms in 2 age groups (younger than 5 years and 5-9 years) and a control group for children aged 5-9 years. Standard SMC (SP-AQ) for children younger than 5 years was provided to the control arm, while SMC with SP-AQ was delivered to children aged 3 months to 9 years (arm 2), and SMC with DHA-PQ will be implemented in study arm 3 for children up to 9 years of age. The study was performed in Mali's Koulikoro District, a rural area in southwest Mali with historically high malaria transmission rates. The study's primary outcome is P falciparum incidence for 2 SMC regimens in children up to 9 years of age. Should DHA-PQ provide an acceptable alternative to SP-AQ, a plausible second-line prevention option would be available in the event of SP resistance or drug supply shortages. A significant byproduct of this effort included bolstering district health information systems for rapid identification of severe malaria cases. RESULTS: The study began on July 1, 2019. Through November 2022, a total of 4556 children 3 months old to younger than 5 years were enrolled. Data collection ended in spring 2023, and the findings are expected to be published later in early 2024. CONCLUSIONS: Routine evaluation of antimalarial drugs is needed to establish appropriate SMC age targets. The study goals here may impact public health policy and provide alternative therapies in the event of drug shortages or resistance. TRIAL REGISTRATION: ClinicalTrials.gov NCT04149106, https://clinicaltrials.gov/ct2/show/NCT04149106. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/51660.

Plasmodium GPI-anchored micronemal antigen is essential for parasite transmission through the mosquito host.

Plasmodium parasites, the eukaryotic pathogens that cause malaria, feature three distinct invasive forms tailored to the host environment they must navigate and invade for life cycle progression. One conserved feature of these invasive forms is the micronemes, apically oriented secretory organelles involved in egress, motility, adhesion, and invasion. Here we investigate the role of GPI-anchored micronemal antigen (GAMA), which shows a micronemal localization in all zoite forms of the rodent-infecting species Plasmodium berghei. ∆GAMA parasites are severely defective for invasion of the mosquito midgut. Once formed, oocysts develop normally, however, sporozoites are unable to egress and exhibit defective motility. Epitope-tagging of GAMA revealed tight temporal expression late during sporogony and showed that GAMA is shed during sporozoite gliding motility in a similar manner to circumsporozoite protein. Complementation of P. berghei knockout parasites with full-length P. falciparum GAMA partially restored infectivity to mosquitoes, indicating conservation of function across Plasmodium species. A suite of parasites with GAMA expressed under the promoters of CTRP, CAP380, and TRAP, further confirmed the involvement of GAMA in midgut infection, motility, and vertebrate infection. These data show GAMA's involvement in sporozoite motility, egress, and invasion, implicating GAMA as a regulator of microneme function.

Molecular markers for malaria genetic epidemiology: progress and pitfalls.

Over recent years, progress in molecular markers for genotyping malaria parasites has enabled informative studies of epidemiology and transmission dynamics. Results have highlighted the value of these tools for surveillance to support malaria control and elimination strategies. There are many different types and panels of markers available for malaria parasite genotyping, and for end users, the nuances of these markers with respect to 'use case', resolution, and accuracy, are not well defined. This review clarifies issues surrounding different molecular markers and their application to malaria control and elimination. We describe available marker panels, use cases, implications for different transmission settings, limitations, access, cost, and data accuracy. The information provided can be used as a guide for molecular epidemiology and surveillance of malaria.

Imported malaria into Australia: surveillance insights and opportunities.

BACKGROUND: Malaria continues to pose a significant burden in endemic countries, many of which lack access to molecular surveillance. Insights from malaria cases in travellers returning to non-endemic areas can provide valuable data to inform endemic country programmes. To evaluate the potential for novel global insights into malaria, we examined epidemiological and molecular data from imported malaria cases to Australia. METHODS: We analysed malaria cases reported in Australia from 2012 to 2022 using National Notifiable Disease Surveillance System data. Molecular data on imported malaria cases were obtained from literature searches. RESULTS: Between 2012 and 2022, 3204 malaria cases were reported in Australia. Most cases (69%) were male and 44% occurred in young adults aged 20-39 years. Incidence rates initially declined between 2012 and 2015, then increased until 2019. During 2012-2019, the incidence in travellers ranged from 1.34 to 7.71 per 100 000 trips. Cases were primarily acquired in Sub-Saharan Africa (n = 1433; 45%), Oceania (n = 569; 18%) and Southern and Central Asia (n = 367; 12%). The most common countries of acquisition were Papua New Guinea (n = 474) and India (n = 277). Plasmodium falciparum accounted for 58% (1871/3204) of cases and was predominantly acquired in Sub-Saharan Africa, and Plasmodium vivax accounted for 32% (1016/3204), predominantly from Oceania and Asia. Molecular studies of imported malaria cases to Australia identified genetic mutations and deletions associated with drug resistance and false-negative rapid diagnostic test results, and led to the establishment of reference genomes for P. vivax and Plasmodium malariae. CONCLUSIONS: Our analysis highlights the continuing burden of imported malaria into Australia. Molecular studies have offered valuable insights into drug resistance and diagnostic limitations, and established reference genomes. Integrating molecular data into national surveillance systems could provide important infectious disease intelligence to optimize treatment guidelines for returning travellers and support endemic country surveillance programmes.

Modeling clinical malaria episodes in different ecological settings in Mali, 2018-2022.

Objectives: Following the scaling-up of malaria control strategies in Mali, understanding the changes in age-specific prevalence of infection and risk factors associated with remains necessary to determine new priorities to progress toward disease elimination. This study aimed to estimate the risk of clinical malaria using longitudinal data across three different transmission settings in Mali. Methods: Cohort-based longitudinal studies were performed from April 2018 to December 2022. Incidence of malaria was measured through community health center-based passive case detection. Generalized estimation equation model was used to assess risk factors for clinical malaria. Results: A total of 21,453 clinical presentations were reported from 4500 participants, mainly from July to November. Data shows a significant association between malaria episodes, sex, age group, season, and year. Women had lower risk, the risk of clinical episode increased with age up to 14 years then declined, and in both sites, the dry-season risk of clinical episode was significantly lower compared to the rainy season. Conclusion: Determining factors associated with the occurrence of clinical malaria across different ecological settings across the country could help in the development of new strategies aiming to accelerate malaria elimination in an area where malaria transmission remains intense.

Nanopore sequencing for malaria molecular surveillance: opportunities and challenges.

Nanopore-based sequencing platforms offer the potential for affordable malaria molecular surveillance (MMS) in resource-limited settings to track and ultimately counteract emerging threats, such as drug resistance and diagnostic escape. Here, we discuss opportunities and challenges to implementing MMS using nanopore sequencing, highlighting priority areas for technical development and innovation.

A Pyridyl-Furan Series Developed from the Open Global Health Library Block Red Blood Cell Invasion and Protein Trafficking in Plasmodium falciparum through Potential Inhibition of the Parasite's PI4KIIIB Enzyme.

With the resistance increasing to current antimalarial medicines, there is an urgent need to discover new drug targets and to develop new medicines against these targets. We therefore screened the Open Global Health Library of Merck KGaA, Darmstadt, Germany, of 250 compounds against the asexual blood stage of the deadliest malarial parasite Plasmodium falciparum, from which eight inhibitors with low micromolar potency were found. Due to its combined potencies against parasite growth and inhibition of red blood cell invasion, the pyridyl-furan compound OGHL250 was prioritized for further optimization. The potency of the series lead compound (WEHI-518) was improved 250-fold to low nanomolar levels against parasite blood-stage growth. Parasites selected for resistance to a related compound, MMV396797, were also resistant to WEHI-518 as well as KDU731, an inhibitor of the phosphatidylinositol kinase PfPI4KIIIB, suggesting that this kinase is the target of the pyridyl-furan series. Inhibition of PfPI4KIIIB blocks multiple stages of the parasite's life cycle and other potent inhibitors are currently under preclinical development. MMV396797-resistant parasites possess an E1316D mutation in PfPKI4IIIB that clusters with known resistance mutations of other inhibitors of the kinase. Building upon earlier studies that showed that PfPI4KIIIB inhibitors block the development of the invasive merozoite parasite stage, we show that members of the pyridyl-furan series also block invasion and/or the conversion of merozoites into ring-stage intracellular parasites through inhibition of protein secretion and export into red blood cells.

Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.

Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

A molecular barcode and web-based data analysis tool to identify imported Plasmodium vivax malaria.

Traditionally, patient travel history has been used to distinguish imported from autochthonous malaria cases, but the dormant liver stages of Plasmodium vivax confound this approach. Molecular tools offer an alternative method to identify, and map imported cases. Using machine learning approaches incorporating hierarchical fixation index and decision tree analyses applied to 799 P. vivax genomes from 21 countries, we identified 33-SNP, 50-SNP and 55-SNP barcodes (GEO33, GEO50 and GEO55), with high capacity to predict the infection's country of origin. The Matthews correlation coefficient (MCC) for an existing, commonly applied 38-SNP barcode (BR38) exceeded 0.80 in 62% countries. The GEO panels outperformed BR38, with median MCCs > 0.80 in 90% countries at GEO33, and 95% at GEO50 and GEO55. An online, open-access, likelihood-based classifier framework was established to support data analysis (vivaxGEN-geo). The SNP selection and classifier methods can be readily amended for other use cases to support malaria control programs.

The West Africa ICEMR Partnerships for Guiding Policy to Improve the Malaria Prevention and Control.

The Mali National Malaria Control Program (NMCP) recently established a phased set of goals for eliminating malaria in Mali by 2030. Over the past decade, the scale-up of NMCP-led malaria control interventions has led to considerable progress, as evidenced by multiple malariometric indicators. The West Africa International Center of Excellence in Malaria Research (WA-ICEMR) is a multidisciplinary research program that works closely with the NMCP and its partners to address critical research needs for malaria control. This coordinated effort includes assessing the effectiveness of control interventions based on key malaria research topics, including immune status, parasite genetic diversity, insecticide and drug resistance, diagnostic accuracy, malaria vector populations and biting behaviors, and vectorial capacity. Several signature accomplishments of the WA-ICEMR include identifying changing malaria age demographic profiles, testing innovative approaches to improve control strategies, and providing regular reporting on drug and insecticide resistance status. The NMCP and WA-ICEMR partnership between the WA-ICEMR and the NMCP offers a comprehensive research platform that informs the design and implementation of malaria prevention and control research programs. These efforts build local expertise and capacity for the next generation of malaria researchers and guide local policy, which is crucial in sustaining efforts toward eliminating malaria in West Africa.

Asia-Pacific ICEMR: Understanding Malaria Transmission to Accelerate Malaria Elimination in the Asia Pacific Region.

Gaining an in-depth understanding of malaria transmission requires integrated, multifaceted research approaches. The Asia-Pacific International Center of Excellence in Malaria Research (ICEMR) is applying specifically developed molecular and immunological assays, in-depth entomological assessments, and advanced statistical and mathematical modeling approaches to a rich series of longitudinal cohort and cross-sectional studies in Papua New Guinea and Cambodia. This is revealing both the essential contribution of forest-based transmission and the particular challenges posed by Plasmodium vivax to malaria elimination in Cambodia. In Papua New Guinea, these studies document the complex host-vector-parasite interactions that are underlying both the stunning reductions in malaria burden from 2006 to 2014 and the significant resurgence in transmission in 2016 to 2018. Here we describe the novel analytical, surveillance, molecular, and immunological tools that are being applied in our ongoing Asia-Pacific ICEMR research program.

A Decade of Progress Accelerating Malaria Control in Mali: Evidence from the West Africa International Center of Excellence for Malaria Research.

This article highlights over a decade of signature achievements by the West Africa International Centers for Excellence in Malaria Research (WA-ICEMR) and its partners toward guiding malaria prevention and control strategies. Since 2010, the WA-ICEMR has performed longitudinal studies to monitor and assess malaria control interventions with respect to space-time patterns, vector transmission indicators, and drug resistance markers. These activities were facilitated and supported by the Mali National Malaria Control Program. Research activities included large-scale active and passive surveillance and expanded coverage of universal long-lasting insecticide-treated bed nets and seasonal malaria chemoprevention (SMC). The findings revealed substantial declines in malaria occurrence after the scale-up of control interventions in WA-ICEMR study sites. WA-ICEMR studies showed that SMC using sulfadoxine-pyrimethamine plus amodiaquine was highly effective in preventing malaria among children under 5 years of age. An alternative SMC regimen (dihydroartemisinin plus piperaquine) was shown to be potentially more effective and provided advantages for acceptability and compliance over the standard SMC regimen. Other findings discussed in this article include higher observed multiplicity of infection rates for malaria in historically high-endemic areas, continued antimalarial drug sensitivity to Plasmodium falciparum, high outdoor malaria transmission rates, and increased insecticide resistance over the past decade. The progress achieved by the WA-ICEMR and its partners highlights the critical need for maintaining current malaria control interventions while developing novel strategies to disrupt malaria transmission. Enhanced evaluation of these strategies through research partnerships is particularly needed in the wake of reported artemisinin resistance in Southeast Asia and East Africa.

VIVID: A Web Application for Variant Interpretation and Visualization in Multi-dimensional Analyses.

Large-scale comparative genomics- and population genetic studies generate enormous amounts of polymorphism data in the form of DNA variants. Ultimately, the goal of many of these studies is to associate genetic variants to phenotypes or fitness. We introduce VIVID, an interactive, user-friendly web application that integrates a wide range of approaches for encoding genotypic to phenotypic information in any organism or disease, from an individual or population, in three-dimensional (3D) space. It allows mutation mapping and annotation, calculation of interactions and conservation scores, prediction of harmful effects, analysis of diversity and selection, and 3D visualization of genotypic information encoded in Variant Call Format on AlphaFold2 protein models. VIVID enables the rapid assessment of genes of interest in the study of adaptive evolution and the genetic load, and it helps prioritizing targets for experimental validation. We demonstrate the utility of VIVID by exploring the evolutionary genetics of the parasitic protist Plasmodium falciparum, revealing geographic variation in the signature of balancing selection in potential targets of functional antibodies.

An open dataset of Plasmodium vivax genome variation in 1,895 worldwide samples.

This report describes the MalariaGEN Pv4 dataset, a new release of curated genome variation data on 1,895 samples of Plasmodium vivax collected at 88 worldwide locations between 2001 and 2017. It includes 1,370 new samples contributed by MalariaGEN and VivaxGEN partner studies in addition to previously published samples from these and other sources. We provide genotype calls at over 4.5 million variable positions including over 3 million single nucleotide polymorphisms (SNPs), as well as short indels and tandem duplications. This enlarged dataset highlights major compartments of parasite population structure, with clear differentiation between Africa, Latin America, Oceania, Western Asia and different parts of Southeast Asia. Each sample has been classified for drug resistance to sulfadoxine, pyrimethamine and mefloquine based on known markers at the dhfr, dhps and mdr1 loci. The prevalence of all of these resistance markers was much higher in Southeast Asia and Oceania than elsewhere. This open resource of analysis-ready genome variation data from the MalariaGEN and VivaxGEN networks is driven by our collective goal to advance research into the complex biology of P. vivax and to accelerate genomic surveillance for malaria control and elimination.

STRIVE PNG: using a partnership-based approach in implementation research to strengthen surveillance and health systems in Papua New Guinea.

Successful implementation research requires effective and equitable relationships between policy-makers, researchers and implementers to effect evidence-based systems change. However, mainstream research grant models between Global North and Global South institutions often (unintentionally) reinforce power imbalances between partners, which result in missed opportunities for knowledge and learning exchange between policy-makers, researchers and implementers.This case study, centred on the STRIVE PNG project, describes how a partnership-based approach has been used to establish, maintain and review effective and equitable relationships between 13 partner organizations (independent research institutes, government health agencies and public health laboratories) to strengthen surveillance and health systems in Papua New Guinea (PNG). We provide an overview of key terms (with supporting conceptual frameworks), describe selected partnership processes and tools used within the project, and share observations regarding early outcomes achieved through this approach.

Global diversity and balancing selection of 23 leading Plasmodium falciparum candidate vaccine antigens.

Investigation of the diversity of malaria parasite antigens can help prioritize and validate them as vaccine candidates and identify the most common variants for inclusion in vaccine formulations. Studies of vaccine candidates of the most virulent human malaria parasite, Plasmodium falciparum, have focused on a handful of well-known antigens, while several others have never been studied. Here we examine the global diversity and population structure of leading vaccine candidate antigens of P. falciparum using the MalariaGEN Pf3K (version 5.1) resource, comprising more than 2600 genomes from 15 malaria endemic countries. A stringent variant calling pipeline was used to extract high quality antigen gene 'haplotypes' from the global dataset and a new R-package named VaxPack was used to streamline population genetic analyses. In addition, a newly developed algorithm that enables spatial averaging of selection pressure on 3D protein structures was applied to the dataset. We analysed the genes encoding 23 leading and novel candidate malaria vaccine antigens including csp, trap, eba175, ama1, rh5, and CelTOS. Our analysis shows that current malaria vaccine formulations are based on rare haplotypes and thus may have limited efficacy against natural parasite populations. High levels of diversity with evidence of balancing selection was detected for most of the erythrocytic and pre-erythrocytic antigens. Measures of natural selection were then mapped to 3D protein structures to predict targets of functional antibodies. For some antigens, geographical variation in the intensity and distribution of these signals on the 3D structure suggests adaptation to different human host or mosquito vector populations. This study provides an essential framework for the diversity of P. falciparum antigens to be considered in the design of the next generation of malaria vaccines.

Population-level genome-wide STR discovery and validation for population structure and genetic diversity assessment of Plasmodium species.

Short tandem repeats (STRs) are highly informative genetic markers that have been used extensively in population genetics analysis. They are an important source of genetic diversity and can also have functional impact. Despite the availability of bioinformatic methods that permit large-scale genome-wide genotyping of STRs from whole genome sequencing data, they have not previously been applied to sequencing data from large collections of malaria parasite field samples. Here, we have genotyped STRs using HipSTR in more than 3,000 Plasmodium falciparum and 174 Plasmodium vivax published whole-genome sequence data from samples collected across the globe. High levels of noise and variability in the resultant callset necessitated the development of a novel method for quality control of STR genotype calls. A set of high-quality STR loci (6,768 from P. falciparum and 3,496 from P. vivax) were used to study Plasmodium genetic diversity, population structures and genomic signatures of selection and these were compared to genome-wide single nucleotide polymorphism (SNP) genotyping data. In addition, the genome-wide information about genetic variation and other characteristics of STRs in P. falciparum and P. vivax have been available in an interactive web-based R Shiny application PlasmoSTR (https://github.com/bahlolab/PlasmoSTR).

Complex infections in vivax malaria: the more you look, the more you find.

The human malaria parasite Plasmodium vivax commonly causes complex multiclonal infections. Recently, Dia et al. have developed innovative methods for single-cell sequencing (SCS) of P. vivax infections by adapting an approach used previously for Plasmodium falciparum. Their studies provide fascinating new insights into P. vivax intrahost diversity and evolution.