Gene expression analyses reveal differences in children's response to malaria according to their age.

In Bandiagara, Mali, children experience on average two clinical malaria episodes per year. However, even in the same transmission area, the number of uncomplicated symptomatic infections, and their parasitemia, can vary dramatically among children. We simultaneously characterize host and parasite gene expression profiles from 136 Malian children with symptomatic falciparum malaria and examine differences in the relative proportion of immune cells and parasite stages, as well as in gene expression, associated with infection and or patient characteristics. Parasitemia explains much of the variation in host and parasite gene expression, and infections with higher parasitemia display proportionally more neutrophils and fewer T cells, suggesting parasitemia-dependent neutrophil recruitment and/or T cell extravasation to secondary lymphoid organs. The child's age also strongly correlates with variations in gene expression: Plasmodium falciparum genes associated with age suggest that older children carry more male gametocytes, while variations in host gene expression indicate a stronger innate response in younger children and stronger adaptive response in older children. These analyses highlight the variability in host responses and parasite regulation during P. falciparum symptomatic infections and emphasize the importance of considering the children's age when studying and treating malaria infections.

Gene expression analyses reveal differences in children's response to malaria according to their age.

In Bandiagara, Mali, children experience on average two clinical malaria episodes per season. However, even in the same transmission area, the number of uncomplicated symptomatic infections, and their parasitemia, vary dramatically among children. To examine the factors contributing to these variations, we simultaneously characterized the host and parasite gene expression profiles from 136 children with symptomatic falciparum malaria and analyzed the expression of 9,205 human and 2,484 Plasmodium genes. We used gene expression deconvolution to estimate the relative proportion of immune cells and parasite stages in each sample and to adjust the differential gene expression analyses. Parasitemia explained much of the variation in both host and parasite gene expression and revealed that infections with higher parasitemia had more neutrophils and fewer T cells, suggesting parasitemia-dependent neutrophil recruitment and/or T cell extravasation to secondary lymphoid organs. The child's age was also strongly correlated with gene expression variations. Plasmodium falciparum genes associated with age suggested that older children carried more male gametocytes, while host genes associated with age indicated a stronger innate response (through TLR and NLR signaling) in younger children and stronger adaptive immunity (through TCR and BCR signaling) in older children. These analyses highlight the variability in host responses and parasite regulation during P. falciparum symptomatic infections and emphasize the importance of considering the children's age when studying and treating malaria infections.

Gene expression analyses reveal differences in children's response to malaria according to their age.

In Bandiagara, Mali, children experience on average two clinical malaria episodes per season. However, even in the same transmission area, the number of uncomplicated symptomatic infections, and their parasitemia, vary dramatically among children. To examine the factors contributing to these variations, we simultaneously characterized the host and parasite gene expression profiles from 136 children with symptomatic falciparum malaria and analyzed the expression of 9,205 human and 2,484 Plasmodium genes. We used gene expression deconvolution to estimate the relative proportion of immune cells and parasite stages in each sample and to adjust the differential gene expression analyses. Parasitemia explained much of the variation in both host and parasite gene expression and revealed that infections with higher parasitemia had more neutrophils and fewer T cells, suggesting parasitemia-dependent neutrophil recruitment and/or T cell extravasation to secondary lymphoid organs. The child's age was also strongly correlated with gene expression variations. Plasmodium falciparum genes associated with age suggested that older children carried more male gametocytes, while host genes associated with age indicated a stronger innate response (through TLR and NLR signaling) in younger children and stronger adaptive immunity (through TCR and BCR signaling) in older children. These analyses highlight the variability in host responses and parasite regulation during P. falciparum symptomatic infections and emphasize the importance of considering the children's age when studying and treating malaria infections.

Malian children infected with Plasmodium ovale and Plasmodium falciparum display very similar gene expression profiles.

Plasmodium parasites caused 241 million cases of malaria and over 600,000 deaths in 2020. Both P. falciparum and P. ovale are endemic to Mali and cause clinical malaria, with P. falciparum infections typically being more severe. Here, we sequenced RNA from nine pediatric blood samples collected during infections with either P. falciparum or P. ovale, and characterized the host and parasite gene expression profiles. We found that human gene expression varies more between individuals than according to the parasite species causing the infection, while parasite gene expression profiles cluster by species. Additionally, we characterized DNA polymorphisms of the parasites directly from the RNA-seq reads and found comparable levels of genetic diversity in both species, despite dramatic differences in prevalence. Our results provide unique insights into host-pathogen interactions during malaria infections and their variations according to the infecting Plasmodium species, which will be critical to develop better elimination strategies against all human Plasmodium parasites.

Host and Parasite Transcriptomic Changes upon Successive Plasmodium falciparum Infections in Early Childhood.

Children are highly susceptible to clinical malaria, and in regions where malaria is endemic, their immune systems must face successive encounters with Plasmodium falciparum parasites before they develop immunity, first against severe disease and later against uncomplicated malaria. Understanding cellular and molecular interactions between host and parasites during an infection could provide insights into the processes underlying this gradual acquisition of immunity, as well as to how parasites adapt to infect hosts that are successively more malaria experienced. Here, we describe methods to analyze the host and parasite gene expression profiles generated simultaneously from blood samples collected from five consecutive symptomatic P. falciparum infections in three Malian children. We show that the data generated enable statistical assessment of the proportions of (i) each white blood cell subset and (ii) the parasite developmental stages, as well as investigations of host-parasite gene coexpression. We also use the sequences generated to analyze allelic variations in transcribed regions and determine the complexity of each infection. While limited by the modest sample size, our analyses suggest that host gene expression profiles primarily clustered by individual, while the parasite gene expression profiles seemed to differentiate early from late infections. Overall, this study provides a solid framework to examine the mechanisms underlying acquisition of immunity to malaria infections using whole-blood transcriptome sequencing (RNA-seq).IMPORTANCE We show that dual RNA-seq from patient blood samples allows characterization of host/parasite interactions during malaria infections and can provide a solid framework to study the acquisition of antimalarial immunity, as well as the adaptations of P. falciparum to malaria-experienced hosts.

New var reconstruction algorithm exposes high var sequence diversity in a single geographic location in Mali.

BACKGROUND: Encoded by the var gene family, highly variable Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) proteins mediate tissue-specific cytoadherence of infected erythrocytes, resulting in immune evasion and severe malaria disease. Sequencing and assembling the 40-60 var gene complement for individual infections has been notoriously difficult, impeding molecular epidemiological studies and the assessment of particular var elements as subunit vaccine candidates. METHODS: We developed and validated a novel algorithm, Exon-Targeted Hybrid Assembly (ETHA), to perform targeted assembly of var gene sequences, based on a combination of Pacific Biosciences and Illumina data. RESULTS: Using ETHA, we characterized the repertoire of var genes in 12 samples from uncomplicated malaria infections in children from a single Malian village and showed them to be as genetically diverse as vars from isolates from around the globe. The gene var2csa, a member of the var family associated with placental malaria pathogenesis, was present in each genome, as were vars previously associated with severe malaria. CONCLUSION: ETHA, a tool to discover novel var sequences from clinical samples, will aid the understanding of malaria pathogenesis and inform the design of malaria vaccines based on PfEMP1. ETHA is available at: https://sourceforge.net/projects/etha/ .

Hemoglobin C Trait Provides Protection From Clinical Falciparum Malaria in Malian Children.

BACKGROUND: Hemoglobin C trait, like hemoglobin S trait, protects against severe malaria in children, but it is unclear whether hemoglobin C trait also protects against uncomplicated malaria. We hypothesized that Malian children with hemoglobin C trait would have a lower risk of clinical malaria than children with hemoglobin AA. METHODS: Three hundred children aged 0-6 years were enrolled in a cohort study of malaria incidence in Bandiagara, Mali, with continuous passive and monthly active follow-up from June 2009 to June 2010. RESULTS: Compared to hemoglobin AA children (n = 242), hemoglobin AC children (n = 39) had a longer time to first clinical malaria episode (hazard ratio [HR], 0.19; P = .001; 364 median malaria-free days vs 181 days), fewer episodes of clinical malaria, and a lower cumulative parasite burden. Similarly, hemoglobin AS children (n = 14) had a longer time to first clinical malaria episode than hemoglobin AA children (HR, 0.15; P = .015; 364 median malaria-free days vs 181 days), but experienced the most asymptomatic malaria infections of any group. CONCLUSIONS: Both hemoglobin C and S traits exerted a protective effect against clinical malaria episodes, but appeared to do so by mechanisms that differentially affect the response to infecting malaria parasites.

Stable malaria incidence despite scaling up control strategies in a malaria vaccine-testing site in Mali.

BACKGROUND: The recent decline in malaria incidence in many African countries has been attributed to the provision of prompt and effective anti-malarial treatment using artemisinin-based combination therapy (ACT) and to the widespread distribution of long-lasting, insecticide-treated bed nets (LLINs). At a malaria vaccine-testing site in Bandiagara, Mali, ACT was introduced in 2004, and LLINs have been distributed free of charge since 2007 to infants after they complete the Expanded Programme of Immunization (EPI) schedule and to pregnant women receiving antenatal care. These strategies may have an impact on malaria incidence. METHODS: To document malaria incidence, a cohort of 400 children aged 0 to 14 years was followed for three to four years up to July 2013. Monthly cross-sectional surveys were done to measure the prevalence of malaria infection and anaemia. Clinical disease was measured both actively and passively through continuous availability of primary medical care. Measured outcomes included asymptomatic Plasmodium infection, anaemia and clinical malaria episodes. RESULTS: The incidence rate of clinical malaria varied significantly from June 2009 to July 2013 without a clear downward trend. A sharp seasonality in malaria illness incidence was observed with higher clinical malaria incidence rates during the rainy season. Parasite and anaemia point prevalence also showed seasonal variation with much higher prevalence rates during rainy seasons compared to dry seasons. CONCLUSIONS: Despite the scaling up of malaria prevention and treatment, including the widespread use of bed nets, better diagnosis and wider availability of ACT, malaria incidence did not decrease in Bandiagara during the study period.

Spatio-temporal analysis of malaria within a transmission season in Bandiagara, Mali.

BACKGROUND: Heterogeneous patterns of malaria transmission are thought to be driven by factors including host genetics, distance to mosquito breeding sites, housing construction, and socio-behavioural characteristics. Evaluation of local transmission epidemiology to characterize malaria risk is essential for planning malaria control and elimination programmes. The use of geographical information systems (GIS) techniques has been a major asset to this approach. To assess time and space distribution of malaria disease in Bandiagara, Mali, within a transmission season, data were used from an ongoing malaria incidence study that enrolled 300 participants aged under six years old". METHODS: Children's households were georeferenced using a handheld global position system. Clinical malaria was defined as a positive blood slide for Plasmodium falciparum asexual stages associated with at least one of the following signs: headache, body aches, fever, chills and weakness. Daily rainfall was measured at the local weather station.Landscape features of Bandiagara were obtained from satellite images and field survey. QGIS™ software was used to map malaria cases, affected and non-affected children, and the number of malaria episodes per child in each block of Bandiagara. Clusters of high or low risk were identified under SaTScan(®) software according to a Bernoulli model. RESULTS: From June 2009 to May 2010, 296 clinical malaria cases were recorded. Though clearly temporally related to the rains, Plasmodium falciparum occurrence persisted late in the dry season. Two "hot spots" of malaria transmission also found, notably along the Yamé River, characterized by higher than expected numbers of malaria cases, and high numbers of clinical episodes per child. Conversely, the north-eastern sector of the town had fewer cases despite its proximity to a large body of standing water which was mosquito habitat. CONCLUSION: These results confirm the existence of a marked spatial heterogeneity of malaria transmission in Bandiagara, providing support for implementation of targeted interventions.