Structural organization of erythrocyte membrane microdomains and their relation with malaria susceptibility.

Cholesterol-rich microdomains are membrane compartments characterized by specific lipid and protein composition. These dynamic assemblies are involved in several biological processes, including infection by intracellular pathogens. This work provides a comprehensive analysis of the composition of human erythrocyte membrane microdomains. Based on their floating properties, we also categorized the microdomain-associated proteins into clusters. Interestingly, erythrocyte microdomains include the vast majority of the proteins known to be involved in invasion by the malaria parasite Plasmodium falciparum. We show here that the Ecto-ADP-ribosyltransferase 4 (ART4) and Aquaporin 1 (AQP1), found within one specific cluster, containing the essential host determinant CD55, are recruited to the site of parasite entry and then internalized to the newly formed parasitophorous vacuole membrane. By generating null erythroid cell lines, we showed that one of these proteins, ART4, plays a role in P. falciparum invasion. We also found that genetic variants in both ART4 and AQP1 are associated with susceptibility to the disease in a malaria-endemic population.

Modulation of Malaria Phenotypes by Pyruvate Kinase (PKLR) Variants in a Thai Population.

Pyruvate kinase (PKLR) is a critical erythrocyte enzyme that is required for glycolysis and production of ATP. We have shown that Pklr deficiency in mice reduces the severity (reduced parasitemia, increased survival) of blood stage malaria induced by infection with Plasmodium chabaudi AS. Likewise, studies in human erythrocytes infected ex vivo with P. falciparum show that presence of host PK-deficiency alleles reduces infection phenotypes. We have characterized the genetic diversity of the PKLR gene, including haplotype structure and presence of rare coding variants in two populations from malaria endemic areas of Thailand and Senegal. We investigated the effect of PKLR genotypes on rich longitudinal datasets including haematological and malaria-associated phenotypes. A coding and possibly damaging variant (R41Q) was identified in the Thai population with a minor allele frequency of ~4.7%. Arginine 41 (R41) is highly conserved in the pyruvate kinase family and its substitution to Glutamine (R41Q) affects protein stability. Heterozygosity for R41Q is shown to be associated with a significant reduction in the number of attacks with Plasmodium falciparum, while correlating with an increased number of Plasmodium vivax infections. These results strongly suggest that PKLR protein variants may affect the frequency, and the intensity of malaria episodes induced by different Plasmodium parasites in humans living in areas of endemic malaria.

Should malaria treatment be guided by a point of care rapid test? A threshold approach to malaria management in rural Burkina Faso.

BACKGROUND: In Burkina Faso, rapid diagnostic tests for malaria have been made recently available. Previously, malaria was managed clinically. This study aims at assessing which is the best management option of a febrile patient in a hyperendemic setting. Three alternatives are: treating presumptively, testing, or refraining from both test and treatment. The test threshold is the tradeoff between refraining and testing, the test-treatment threshold is the tradeoff between testing and treating. Only if the disease probability lies between the two should the test be used. METHODS AND FINDINGS: Data for this analysis was obtained from previous studies on malaria rapid tests, involving 5220 patients. The thresholds were calculated, based on disease risk, treatment risk and cost, test accuracy and cost. The thresholds were then matched against the disease probability. For a febrile child under 5 in the dry season, the pre-test probability of clinical malaria (3.2%), was just above the test/treatment threshold. In the rainy season, that probability was 63%, largely above the test/treatment threshold. For febrile children >5 years and adults in the dry season, the probability was 1.7%, below the test threshold, while in the rainy season it was higher (25.1%), and situated between the two thresholds (3% and 60.9%), only if costs were not considered. If they were, neither testing nor treating with artemisinin combination treatments (ACT) would be recommended. CONCLUSIONS: A febrile child under 5 should be treated presumptively. In the dry season, the probability of clinical malaria in adults is so low, that neither testing nor treating with any regimen should be recommended. In the rainy season, if costs are considered, a febrile adult should not be tested, nor treated with ACT, but a possible alternative would be a presumptive treatment with amodiaquine plus sulfadoxine-pyrimethamine. If costs were not considered, testing would be recommended.

Rapid malaria diagnostic tests vs. clinical management of malaria in rural Burkina Faso: safety and effect on clinical decisions. A randomized trial.

OBJECTIVES: To assess if the clinical outcome of patients treated after performing a Rapid Diagnostic Test for malaria (RDT) is at least equivalent to that of controls (treated presumptively without test) and to determine the impact of the introduction of a malaria RDT on clinical decisions. METHODS: Randomized, multi-centre, open clinical trial in two arms in 2006 at the end of the dry and of the rainy season in 10 peripheral health centres in Burkina Faso: one arm with use of RDT before treatment decision, one arm managed clinically. Primary endpoint: persistence of fever at day 4. Secondary endpoints: frequency of malaria treatment and of antibiotic treatment. RESULTS: A total of 852 febrile patients were recruited in the dry season and 1317 febrile patients in the rainy season, and randomized either to be submitted to RDT (P_RTD) or to be managed presumptively (P_CLIN). In both seasons, no significant difference was found between the two randomized groups in the frequency of antimalarial treatment, nor of antibiotic prescription. In the dry season, 80.8% and 79.8% of patients with a negative RDT were nevertheless diagnosed and treated for malaria, and so were 85.0% and 82.6% negative patients in the rainy season. In the rainy season only, both diagnosis and treatment of other conditions were significantly less frequent in RDT positive vs. negative patients (48.3% vs. 61.4% and 46.2% vs. 59.9%, P = 0.00 and 0.00, respectively). CONCLUSION: Our study was inconclusive on RDT safety (clinical outcome in the two randomized groups), because of an exceedingly and unexpectedly low compliance with the negative test result. Further research is needed on best strategies to promote adherence and on the safety of a test based strategy compared with the current, presumptive treatment strategy.

Functional deficit of T regulatory cells in Fulani, an ethnic group with low susceptibility to Plasmodium falciparum malaria.

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, IFNgamma, 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 TGFbeta, TGFbetaRs, 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 TGFbeta 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.

IL4-589C/T polymorphism and IgE levels in severe malaria.

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.

IgE antibodies to Plasmodium falciparum and severity of malaria in children of one ethnic group living in Burkina Faso.

Plasmodium falciparum malaria infection induces elevated blood levels of both total immunoglobulin and anti-plasmodial antibodies belonging to different isotypes. We have previously shown that donors living in areas of malaria transmission develop malaria-specific IgE antibodies that are present at highest concentrations in patients with severe disease, suggesting a role for this isotype in malaria pathogenesis. To establish the possible importance of IgE in the course and severity of this disease, we have analyzed a large and homogenous group of African children (age range = 6 months to 15 years) belonging to one ethnic group (Mossi) living in identical epidemiologic conditions in the same urban area (Ougadougo) of Burkina Faso. While IgG antibodies to P. falciparum increased to high concentrations in very young children and then remained at these levels in older patients, IgE antibodies increased with age, becoming most significantly elevated in children more than four years of age. In older children, those with severe malaria had significantly higher IgE antibody levels than those with non-severe disease. No significant differences between the patient groups were seen for IgG antibodies to P. falciparum. However, when the patients with severe malaria were divided into two groups distinguished by the presence of absence of coma, both IgG and IgE antibodies against malaria were lower in the comatous patients than in the non-comatous patients. The results support the conclusion that IgE antibodies against malaria, regardless of their possible protectivity, also contribute to disease severity in this large and homogenous group of African children.