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Host genetic factors can confer resistance against malaria 1 , raising the question of whether this has led to evolutionary adaptation of parasite populations. Here we searched for association between candidate host and parasite genetic variants in 3,346 Gambian and Kenyan children with severe malaria caused by Plasmodium falciparum. We identified a strong association between sickle haemoglobin (HbS) in the host and three regions of the parasite genome, which is not explained by population structure or other covariates, and which is replicated in additional samples. The HbS-associated alleles include nonsynonymous variants in the gene for the acyl-CoA synthetase family member 2-4 PfACS8 on chromosome 2, in a second region of chromosome 2, and in a region containing structural variation on chromosome 11. The alleles are in strong linkage disequilibrium and have frequencies that covary with the frequency of HbS across populations, in particular being much more common in Africa than other parts of the world. The estimated protective effect of HbS against severe malaria, as determined by comparison of cases with population controls, varies greatly according to the parasite genotype at these three loci. These findings open up a new avenue of enquiry into the biological and epidemiological significance of the HbS-associated polymorphisms in the parasite genome and the evolutionary forces that have led to their high frequency and strong linkage disequilibrium in African P. falciparum populations., (© 2021. The Author(s).)

Background: Cerebral malaria (CM) is a severe neurological complication of Plasmodium falciparum infection. A number of pathological findings have been correlated with pediatric CM including sequestration, platelet accumulation, petechial haemorrhage and retinopathy. However, the molecular mechanisms leading to death in CM are not yet fully understood., Methods: A shotgun plasma proteomic study was conducted using samples form 52 Gambian children with CM admitted to hospital. Based on clinical outcome, children were assigned to two groups: reversible and fatal CM. Label-free liquid chromatography-tandem mass spectrometry was used to identify and compare plasma proteins that were differentially regulated in children who recovered from CM and those who died. Candidate biomarkers were validated using enzyme immunoassays., Results: The plasma proteomic signature of children with CM identified 266 proteins differentially regulated in children with fatal CM. Proteins from the coagulation cascade were consistently decreased in fatal CM, whereas the plasma proteomic signature associated with fatal CM underscored the importance of endothelial activation, tissue damage, inflammation, haemolysis and glucose metabolism. The concentration of circulating proteasomes or PSMB9 in plasma was not significantly different in fatal CM when compared with survivors. Plasma PSMB9 concentration was higher in patients who presented with seizures and was significantly correlated with the number of seizures observed in patients with CM during admission., Conclusions: The results indicate that increased tissue damage and hypercoagulability may play an important role in fatal CM. The diagnostic value of this molecular signature to identify children at high risk of dying to optimize patient referral practices should be validated prospectively.

The relevance of innate immune responses to Plasmodium falciparum infection, in particular the central role of natural killer (NK) cell-derived interferon gamma (IFN-γ), is becoming increasingly recognised. Recently, it has been shown that IFN-γ production in response to P. falciparum antigens is in part regulated by killer-cell immunoglobulin-like receptor (KIR) genes, and a study from malaria-exposed Melanesians suggested an association between KIR genotypes and susceptibility to infection. This prompted us to determine and compare the frequencies of 15 KIR genes in Gambian children presenting with either severe malaria (n = 133) or uncomplicated malaria (n = 188) and in cord-blood population control samples (n = 314) collected from the same area. While no significant differences were observed between severe and uncomplicated cases, proportions of individuals with KIR2DS2+C1 and KIR2DL2+C1 were significantly higher among malaria cases overall than in population control samples. In an exploratory analysis, activating KIR genes KIR2DS2, KIR3DS1 and KIR2DS5 were slightly higher in children in disease subgroups associated with the highest mortality. In addition, our data suggest that homozygosity for KIR genotype A might be associated with different malaria outcomes including protection from infection and higher blood parasitaemia levels in those that do get infected. These findings are consistent with a probable role of KIR genes in determining susceptibility to malaria, and further studies are warranted in different populations., (© 2012 John Wiley & Sons A/S.)

Acquired immunity in vertebrates maintains polymorphisms in endemic pathogens, leading to identifiable signatures of balancing selection. To comprehensively survey for genes under such selection in the human malaria parasite Plasmodium falciparum, we generated paired-end short-read sequences of parasites in clinical isolates from an endemic Gambian population, which were mapped to the 3D7 strain reference genome to yield high-quality genome-wide coding sequence data for 65 isolates. A minority of genes did not map reliably, including the hypervariable var, rifin, and stevor families, but 5,056 genes (90.9% of all in the genome) had >70% sequence coverage with minimum read depth of 5 for at least 50 isolates, of which 2,853 genes contained 3 or more single nucleotide polymorphisms (SNPs) for analysis of polymorphic site frequency spectra. Against an overall background of negatively skewed frequencies, as expected from historical population expansion combined with purifying selection, the outlying minority of genes with signatures indicating exceptionally intermediate frequencies were identified. Comparing genes with different stage-specificity, such signatures were most common in those with peak expression at the merozoite stage that invades erythrocytes. Members of clag, PfMC-2TM, surfin, and msp3-like gene families were highly represented, the strongest signature being in the msp3-like gene PF10&#95;0355. Analysis of msp3-like transcripts in 45 clinical and 11 laboratory adapted isolates grown to merozoite-containing schizont stages revealed surprisingly low expression of PF10&#95;0355. In diverse clonal parasite lines the protein product was expressed in a minority of mature schizonts (<1% in most lines and ∼10% in clone HB3), and eight sub-clones of HB3 cultured separately had an intermediate spectrum of positive frequencies (0.9 to 7.5%), indicating phase variable expression of this polymorphic antigen. This and other identified targets of balancing selection are now prioritized for functional study., Competing Interests: The authors have declared that no competing interests exist.

Background: During malaria infection the Toll-like receptor 9 (TLR9) is activated through induction with plasmodium DNA or another malaria motif not yet identified. Although TLR9 activation by malaria parasites is well reported, the implication to the susceptibility to severe malaria is not clear. The aim of this study was to assess the contribution of genetic variation at TLR9 to severe malaria., Methods: This study explores the contribution of TLR9 genetic variants to severe malaria using two approaches. First, an association study of four common single nucleotide polymorphisms was performed on both family- and population-based studies from Malawian and Gambian populations (n>6000 individual). Subsequently, it was assessed whether TLR9 expression is affected by cis-acting variants and if these variants could be mapped. For this work, an allele specific expression (ASE) assay on a panel of HapMap cell lines was carried out., Results: No convincing association was found with polymorphisms in TLR9 for malaria severity, in either Gambian or Malawian populations, using both case-control and family based study designs. Using an allele specific expression assay it was observed that TLR9 expression is affected by cis-acting variants, these results were replicated in a second experiment using biological replicates., Conclusion: By using the largest cohorts analysed to date, as well as a standardized phenotype definition and study design, no association of TLR9 genetic variants with severe malaria was found. This analysis considered all common variants in the region, but it is remains possible that there are rare variants with association signals. This report also shows that TLR9 expression is potentially modulated through cis-regulatory variants, which may lead to differential inflammatory responses to infection between individuals.

There has been much debate about the relative merits of population- and family-based strategies for testing genetic association, yet there is little empirical data that directly compare the two approaches. Here we compare case-control and transmission/disequilibrium test (TDT) study designs using a well-established genetic association, the protective effect of the sickle-cell trait against severe malaria. We find that the two methods give similar estimates of the level of protection (case-control odds ratio = 0.10, 95% confidence interval 0.03–0.23; family-based estimate of the odds ratio = 0.11, 95% confidence interval 0.04–0.25) and similar statistical significance of the result (case-control: , TDT: ) when 315 TDT cases are compared to 583 controls. We propose a family plus population control study design, which allows both case-control and TDT analysis of the cases. This combination is robust against the respective weaknesses of the case-control and TDT study designs, namely population structure and segregation distortion. The combined study design is especially cost-effective when cases are difficult to ascertain and, when the case-control and TDT results agree, offers greater confidence in the result. [ABSTRACT FROM AUTHOR]