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MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Background: The estimation of Plasmodium falciparum parasitaemia can vary according to the method used. Recently, droplet digital PCR (ddPCR) has been proposed as a promising approach in the molecular quantitation of Plasmodium, but its ability to predict the actual parasitaemia on clinical samples has not been largely investigated. Moreover, the possibility of applying the ddPCR-sensitive method to serum samples has never been explored. Methods: We used, for the first time, ddPCR on both blood and serum to detect the DNA of P. falciparum in 52 paired samples from 26 patients. ddPCR was compared with loop-mediated isothermal amplification (LAMP) and rtPCR. The correlation between the ddPCR results, microscopy, and clinical parameters was examined. Results: ddPCR and microscopy were found to be strongly correlated (&rho, (26) = 0.83111, p &lt, 0.0001) in blood. Samples deviating from the correlation were partially explained by clinical parameters. In serum samples, ddPCR revealed the best performance in detecting P. falciparum DNA, with 77% positive samples among malaria subjects. Conclusion: Absolute quantitation by ddPCR can be a flexible technique for Plasmodium detection, with potential application in the diagnosis of malaria. In particular, ddPCR is a powerful approach for Plasmodium DNA analysis on serum when blood samples are unavailable.

Background Transfusion with Plasmodium-infected blood represents a risk for malaria transmission, a rare but severe event. Several non-endemic countries implement a strategy for the screening of candidate blood donors including questionnaire for the identification of at-risk subjects and laboratory testing of blood samples, often serology-based, with temporary deferral from donation for individuals with a positive result. In Italy, the most recent legislation, issued in November 2015, introduced the use of serological tests for the detection of anti-Plasmodium antibodies. Methods In the absence of a gold standard for malaria serology, the aim of this work was to evaluate five commercial ELISA kits, and to determine their accuracy (sensitivity and specificity) in comparison to immuno-fluorescence antibody test (IFAT), and their agreement (concordance of results). Serum samples from malaria patients or from subjects with malaria history (N = 64), malaria naïve patients with other parasitic infections (N = 15), malaria naïve blood donors (N = 8) and malaria exposed candidate blood donors (N = 36) were tested. Results The specificity of all ELISA kits was 100%, while sensitivity ranged between 53 and 64% when compared to IFAT on malaria patients samples. When tested on candidate blood donors’ samples, ELISA kits showed highly variable agreement (42–94%) raising the possibility that the same individual could be included or excluded from donation depending on the test in use by the transfusion centre. Conclusions These preliminary results indicate how the lack of a gold standard for malaria serology must be taken into account in the application and future revision of current legislation. There is need of developing more sensitive serological assays. Moreover, the adoption of a unique serological test at national level is recommended, as well as the development of screening algorithms based on multiple laboratory tests, including molecular assays. Electronic supplementary material The online version of this article (10.1186/s12936-019-2650-0) contains supplementary material, which is available to authorized users.

Background Transfusion-transmitted malaria (TTM) is an accidental Plasmodium infection caused by whole blood or a blood component transfusion from a malaria infected donor to a recipient. Infected blood transfusions directly release malaria parasites in the recipient’s bloodstream triggering the development of high risk complications, and potentially leading to a fatal outcome especially in individuals with no previous exposure to malaria or in immuno-compromised patients. A systematic review was conducted on TTM case reports in non-endemic areas to describe the epidemiological characteristics of blood donors and recipients. Methods Relevant articles were retrieved from Pubmed, EMBASE, Scopus, and LILACS. From each selected study the following data were extracted: study area, gender and age of blood donor and recipient, blood component associated with TTM, Plasmodium species, malaria diagnostic method employed, blood donor screening method, incubation period between the infected transfusion and the onset of clinical symptoms in the recipient, time elapsed between the clinical symptoms and the diagnosis of malaria, infection outcome, country of origin of the blood donor and time of the last potential malaria exposure. Results Plasmodium species were detected in 100 TTM case reports with a different frequency: 45% Plasmodium falciparum, 30% Plasmodium malariae, 16% Plasmodium vivax, 4% Plasmodium ovale, 2% Plasmodium knowlesi, 1% mixed infection P. falciparum/P. malariae. The majority of fatal outcomes (11/45) was caused by P. falciparum whilst the other fatalities occurred in individuals infected by P. malariae (2/30) and P. ovale (1/4). However, non P. falciparum fatalities were not attributed directly to malaria. The incubation time for all Plasmodium species TTM case reports was longer than what expected in natural infections. This difference was statistically significant for P. malariae (p = 0.006). A longer incubation time in the recipient together with a chronic infection at low parasite density of the donor makes P. malariae a subtle but not negligible risk for blood safety aside from P. falciparum. Conclusions TTM risk needs to be taken into account in order to enhance the safety of the blood supply chain from donors to recipients by means of appropriate diagnostic tools.

A number of human genetic polymorphisms are prevalent in tropical populations and appear to offer protection against symptomatic and/or severe malaria. We compared the prevalence of four polymorphisms, the sickle hemoglobin mutation (β globin E6V), the α-thalassemia 3.7kb deletion, glucose-6-phosphate dehydrogenase deficiency caused by the common African variant (G6PD A-), and the CD36 T188G mutation in 1344 individuals residing in districts in eastern (Tororo), south-central (Jinja), and southwestern (Kanungu) Uganda. Genes of interest were amplified, amplicons subjected to mutation-specific restriction endonuclease digestion (for sickle hemoglobin, G6PD A-, and CD36 T188G), reaction products resolved by electrophoresis, and genotypes determined based on the sizes of reaction products. Mutant genotypes were common, with many more heterozygous than homozygous alleles identified. The prevalences (heterozygotes plus homozygotes) of sickle hemoglobin (28% Tororo, 25% Jinja, 7% Kanungu), α-thalassemia (53% Tororo, 45% Jinja, 18% Kanungu) and G6PD A- (29% Tororo, 18% Jinja, 8% Kanungu) were significantly greater in Tororo and Jinja compared to Kanungu (p

The optimal immune response to malaria infection comprises rapid induction of inflammatory responses promptly counteracted by regulatory mechanisms to prevent immunopathology. To evaluate the role of dendritic cells (DC) in the balance of parasite-induced inflammatory/anti-inflammatory mechanisms, we studied the activity of monocyte-derived dendritic cells (MDDC), previously exposed to soluble extracts of Plasmodium falciparum -infected red blood cells ( Pf SE), in the differentiation of CD4 cells isolated from donors never exposed to malaria infection. We show that MDDC exposed to Pf SE are extremely efficient to induce a contemporary differentiation of TH1 effector cells and T regulatory (Treg) cells in CD4 T cells even when exposed to low concentrations of parasitic extracts. Treg cells induced by MDDC infected with Pf SE (MDDC- Pf SE) produce transforming growth factor beta (TGF-β) and interleukin 10 (IL-10) and are endowed with strong suppressive properties. They also show phenotypical and functional peculiarities, such as the contemporary expression of markers of Treg and TH1 differentiation and higher sensitivity to TLR4 ligands both inducing an increasing production of suppressive cytokines. On the whole, our data indicate that MDDC exposed to Pf SE orchestrate a well-balanced immune response with timely differentiation of TH1 and Treg cells in CD4 cells from nonimmune donors and suggest that, during the infection, the role of MDCC could be particularly relevant in low-parasitemia conditions.

Salivary proteins injected by blood feeding arthropods into their hosts evoke a saliva-specific humoral response which can be useful to evaluate exposure to bites of disease vectors. However, saliva of hematophagous arthropods is a complex cocktail of bioactive factors and its use in immunoassays can be misleading because of potential cross-reactivity to other antigens. Toward the development of a serological marker of exposure to Afrotropical malaria vectors we expressed the Anopheles gambiae gSG6, a small anopheline-specific salivary protein, and we measured the anti-gSG6 IgG response in individuals from a malaria hyperendemic area of Burkina Faso, West Africa. The gSG6 protein was immunogenic and anti-gSG6 IgG levels and/or prevalence increased in exposed individuals during the malaria transmission/rainy season. Moreover, this response dropped during the intervening low transmission/dry season, suggesting it is sensitive enough to detect variation in vector density. Members of the Fulani ethnic group showed higher anti-gSG6 IgG response as compared to Mossi, a result consistent with the stronger immune reactivity reported in this group. Remarkably, anti-gSG6 IgG levels among responders were high in children and gradually declined with age. This unusual pattern, opposite to the one observed with Plasmodium antigens, is compatible with a progressive desensitization to mosquito saliva and may be linked to the continued exposure to bites of anopheline mosquitoes. Overall, the humoral anti-gSG6 IgG response appears a reliable serological indicator of exposure to bites of the main African malaria vectors (An. gambiae, Anopheles arabiensis and, possibly, Anopheles funestus) and it may be exploited for malaria epidemiological studies, development of risk maps and evaluation of anti-vector measures. In addition, the gSG6 protein may represent a powerful model system to get a deeper understanding of molecular and cellular mechanisms underlying the immune tolerance and progressive desensitization to insect salivary allergens.

Phylogenetic analyses of the mitochondrial cytochrome b (cytb), apicoplast caseinolytic protease C (clpC), and 18S rRNA sequences of Plasmodium isolates from chimpanzees along with those of the virulent human malaria parasite P. falciparum showed that the common chimpanzee (Pan troglodytes) malaria parasites, assigned by Rich et al. (2009; Proc. Natl. Acad. Sci. USA 106, 14902–14907) to P. reichenowi, constitute a paraphyletic assemblage. The assumption that P. falciparum diverged from P. reichenowi as recently as 5,000–50,000 years ago would require a rate of synonymous substitution/site/year in cytb and clpC on the order of 10−5–10−6, several orders of magnitude higher than any known from eukaryotic organelle genomes, and would imply an unrealistically recent timing of the most recent common ancestor of P. falciparum mitochondrial genomes. The available data are thus most consistent with the hypothesis that P. reichenowi (in the strict sense) and P. falciparum co-speciated with their hosts about 5–7 million years ago.

Plasmodium falciparum represents one of the strongest selective forces on the human genome. This stable and perennial pressure has contributed to the progressive accumulation in the exposed populations of genetic adaptations to malaria. Descriptive genetic epidemiology provides the initial step of a logical procedure of consequential phases spanning from the identification of genes involved in the resistance/susceptibility to diseases, to the determination of the underlying mechanisms and finally to the possible translation of the acquired knowledge in new control tools. In malaria, the rational development of this strategy is traditionally based on complementary interactions of heterogeneous disciplines going from epidemiology to vaccinology passing through genetics, pathogenesis and immunology. New tools including expression profile analysis and genome-wide association studies are recently available to explore the complex interactions of host-parasite co-evolution. Particularly, the combination of genome-wide association studies with large multi-centre initiatives can overcome the limits of previous results due to local population dynamics. Thus, we anticipate substantial advances in the interpretation and validation of the effects of genetic variation on malaria susceptibility, and thereby on molecular mechanisms of protective immune responses and pathogenesis.

Previous interethnic comparative studies on the susceptibility to malaria performed in West Africa showed that Fulani are more resistant to Plasmodium falciparum malaria than are sympatric ethnic groups. This lower susceptibility is not associated to classic malaria-resistance genes, and the analysis of the immune response to P. falciparum sporozoite and blood stage antigens, as well as non-malaria antigens, revealed higher immune reactivity in Fulani. In the present study we compared the expression profile of a panel of genes involved in immune response in peripheral blood mononuclear cells (PBMC) from Fulani and sympatric Mossi from Burkina Faso. An increased expression of T helper 1 (TH1)-related genes (IL-18, IFNγ, and TBX21) and TH2-related genes (IL-4 and GATA3) and a reduced expression of genes distinctive of T regulatory activity (CTLA4 and FOXP3) were observed in Fulani. Microarray analysis on RNA from CD4 + CD25 + (T regulatory) cells, performed with a panel of cDNA probes specific for 96 genes involved in immune modulation, indicated obvious differences between the two ethnic groups with 23% of genes, including TGFβ, TGFβRs, CTLA4, and FOXP3, less expressed in Fulani compared with Mossi and European donors not exposed to malaria. As further indications of a low T regulatory cell activity, Fulani showed lower serum levels of TGFβ and higher concentrations of the proinflammatory chemokines CXCL10 and CCL22 compared with Mossi; moreover, the proliferative response of Fulani to malaria antigens was not affected by the depletion of CD25 + regulatory cells whereas that of Mossi was significantly increased. The results suggest that the higher resistance to malaria of the Fulani could derive from a functional deficit of T regulatory cells.

Previous studies identified an allelic variant of the IL4 promoter region (IL4-589T) that appears to enhance the transcriptional activity of IL4, and is associated with increased IgE levels. Total serum IgE levels are elevated in malaria endemic regions, and higher in children with severe malaria. Here, we investigated the relationship of the IL4-589C/T polymorphism with severity of the disease in a case-control study of severe malaria in Burkina Faso, West Africa. No association between the IL4-589T and severe malaria was observed. No difference in Plasmodium falciparum-specific IgE was detected between severe and uncomplicated malaria patients. Among children with severe malaria, total IgE levels were significantly elevated in those carrying the IL4-589T allele (P = 0.018). In children with uncomplicated malaria, no significant difference was found. These results raise the possibility that there is a relationship between susceptibility to severe malaria, IgE production and genetic variation in the IL4 region, which merits further investigation in other epidemiological settings.

The Fulani are less clinically susceptible and more immunologically responsive to malaria than neighbouring ethnic groups. Here we report that anti-malarial antibody levels show a wide distribution amongst the Fulani themselves, raising the possibility that quantitative analysis within the Fulani may be an efficient way of screening for important genetic factors. The Th2 cytokine interleukin-4 is an obvious candidate: in Fulani, the IL4-524 T allele is at high frequency and is associated with elevated antibody levels against malaria antigens. These data highlight the possibility of combining inter- and intra-ethnic comparisons to characterize critical determinants of malarial immunity in a natural setting.

Genetic divergence between population samples ofOrchis laxiflora and ofO. palustris from various European locations was studied by electrophoretic analysis of 25 enzyme loci. An average genetic distance of DNei = 1.24 was found between the two taxa, with 12 out of 25 loci showing alternative alleles (diagnostic loci). Genetic heterogeneity was observed within bothO. laxiflora andO. palustris, when northern and southeastern populations were compared, being lower in the former taxon (D = 0.06), than in the latter (D = 0.16). Karyologically, 2n = 36 was found for bothO. laxiflora andO. palustris. O. laxiflora andO. palustris produce hybrids, described asO. ×intermedia. Genotype analysis of several sympatric samples showed the presence of hybrid zones, including F1 hybrids and, in low proportions, recombinant classes, putatively assigned to Fn and backcrosses, as well as a few introgressed individuals of both taxa. These data indicate that hybrids are only partially fertile, with a very limited mixing up of the two parental gene pools; this is also shown by the lack of significant lowering of genetic distances when sympatric and allopatric heterospecific samples are compared. Accordingly,O. laxiflora andO. palustris form a syngameon; nevertheless they can be considered as good taxonomic species, with virtually distinct gene pools, which evolve independently. The genetic variability inO. laxiflora andO. palustris is remarkably low (\(\bar H\)e = 0.05 and\(\bar H\)e = 0.02, respectively). In particular, nearly complete absence of polymorphic loci was found inO. palustris from northcentral Europe. Two hypotheses are considered to explain the low genetic variability of this endangered species.

Background Fulani ethnic group individuals are less susceptible than sympatric Mossi ethnic group, in term of malaria infection severity, and differ in antibody production against malaria antigens. The differences in susceptibility to malaria between Fulani and Mossi ethnic groups are thought to be regulated by different genetic backgrounds and offer the opportunity to compare haematological parameters, Tregs and γδT cell profiles in seasonal and stable malaria transmission settings in Burkina Faso. The study was conducted at two different time points i.e. during the high and low malaria transmission period. Results Two cross-sectional surveys were undertaken in adults above 20 years belonging either to the Fulani or the Mossi ethnic groups 1) at the peak of the malaria transmission season and 2) during the middle of the low malaria transmission season. Full blood counts, proportions of Tregs and γδ T cells were measured at both time-points. As previously shown the Fulani and Mossi ethnic groups showed a consistent difference in P. falciparum infection rates and parasite load. Differential white blood cell counts showed that the absolute lymphocyte counts were higher in the Mossi than in the Fulani ethnic group at both time points. While the proportion of CD4+CD25high was higher in the Fulani ethnic group at the peak of malaria transmission season (p = 0.03), no clear pattern emerged for T regulatory cells expressing FoxP3+ and CD127low. However CD3+γδ+ subpopulations were found to be higher in the Fulani compared to the Mossi ethnic group, and this difference was statistically significant at both time-points (p = 0.004 at low transmission season and p = 0.04 at peak of transmission). Conclusion Our findings on regulatory T cell phenotypes suggest an interesting role for immune regulatory mechanisms in response to malaria. The study also suggests that TCRγδ + cells might contribute to the protection against malaria in the Fulani ethnic group involving their reported parasite inhibitory activities.