Search

In vitro and ex vivo cultivation of Plasmodium (P) falciparum has facilitated active research into the malaria parasite toward the quest for basic knowledge and the discovery of effective drug treatments. Such a drug discovery program is currently difficult for P. malariae simply because of the absence of in vitro and ex vivo cultivation system for its asexual blood stages supporting antimalarial evaluation. Despite availability of artemisinin combination therapies effective on P. falciparum, P. malariae is being increasingly detected in malaria endemic countries. P. malariae is responsible for chronic infections and is associated with a high burden of anemia and morbidity. Here, we optimized and adapted ex vivo conditions under which P. malariae can be cultured and used for screening antimalarial drugs. Subsequently, this enabled us to test compounds such as artemether, chloroquine, lumefantrine, and quinine for ex vivo antimalarial activity against P. malariae.

Malaria infections in pregnancy should be treated promptly with safe and efficacious antimalarial drugs to prevent harmful effects on the mother and fetus. To succeed, the Malian has developed NMCP guidelines for the management of malaria cases in pregnant women. The study aimed at the analysis of the prescription of antimalarial drugs based on the Mali's NMCP guidelines. We conducted a cross-sectional study during malaria transmission season from June to August 2020. The sampling concerned all prescriptions for pregnant women containing at least one antimalarial drug. The frequency of prescription of antimalarial drugs was 85%. 132 (74.16%) were preventive treatments and 46 (25.84%) curative treatments. 30 (90.91%) of pregnant women in the first trimester received one dose of Sulfadoxine-Pyrimethamine. 6 (12.5%) received three doses in the third trimester. Of the 46 antimalarial drugs prescribed for the treatment of uncomplicated malaria, 30 (65.22%) were Artemether-lumefantrine (tablet), 10 (21.74%) were Quinine (tablet). 29 (63.04%) were compliant with NMCP guidelines and 17 (36.96%) were not. The non-compliances concerned 3 prescriptions of Artemether-lumefantrine in the first trimester, 3 and 5 prescriptions of Quinine (tablet) in the second and third trimester respectively and at the end 2 and 4 non-compliances respectively for the prescription of injectable dosage forms of Quinine and Artesunate. This study showed a great noncompliance with the Mali's NMCP guidelines in the prescription of antimalarial in pregnant women. Chemoprophylaxis should be prohibited in the first trimester. Keywords: Curative and Preventive Treatment, Malaria in Pregnancy, Malaria Transmission, Mali

Background In 2006, the National Malaria Control Program in Mali recommended artemisinin-based combination therapy as the first-line treatment for uncomplicated malaria. Since the introduction of artemisinin-based combination therapy, few reports are available on the level of resistance of Plasmodium falciparum to the most common anti-malarial drugs in Mali. Methods From 2016 to 2017, we assessed the ex-vivo drug sensitivity of P. falciparum isolates in Kéniéroba, a village located in a rural area of southern Mali. We collected P. falciparum isolates from malaria-infected children living in Kéniéroba. The isolates were tested for ex-vivo sensitivity to commonly used anti-malarial drugs, namely chloroquine, quinine, amodiaquine, mefloquine, lumefantrine, dihydroartermisinin, and piperaquine. We used the 50% inhibitory concentration determination method, which is based on the incorporation of SYBR® Green into the parasite’s genetic material. Results Plasmodium falciparum isolates were found to have a reduced ex-vivo sensitivity to quinine (25.7%), chloroquine (12.2%), amodiaquine (2.7%), and mefloquine (1.3%). In contrast, the isolates were 100% sensitive to lumefantrine, dihydroartermisinin, and piperaquine. A statistically significant correlation was found between 50% inhibitory concentration values of quinine and amodiaquine (r = 0.80; p

Sickle-trait hemoglobin (HbAS) confers nearly complete protection from severe, life-threatening falciparum malaria in African children. Despite this clear protection, the molecular mechanisms by which HbAS confers these protective phenotypes remain incompletely understood. As a forward genetic screen for aberrant parasite transcriptional responses associated with parasite neutralization in HbAS red blood cells (RBCs), we performed comparative transcriptomic analyses of Plasmodium falciparum in normal (HbAA) and HbAS erythrocytes during both in vitro cultivation of reference parasite strains and naturally occurring P. falciparum infections in Malian children with HbAA or HbAS. During in vitro cultivation, parasites matured normally in HbAS RBCs, and the temporal expression was largely unperturbed of the highly ordered transcriptional program that underlies the parasite’s maturation throughout the intraerythrocytic development cycle (IDC). However, differential expression analysis identified hundreds of transcripts aberrantly expressed in HbAS, largely occurring late in the IDC. Surprisingly, transcripts encoding members of the Maurer’s clefts were overexpressed in HbAS despite impaired parasite protein export in these RBCs, while parasites in HbAS RBCs underexpressed transcripts associated with the endoplasmic reticulum and those encoding serine repeat antigen proteases that promote parasite egress. Analyses of P. falciparum transcriptomes from 32 children with uncomplicated malaria identified stage-specific differential expression: among infections composed of ring-stage parasites, only cyclophilin 19B was underexpressed in children with HbAS, while trophozoite-stage infections identified a range of differentially expressed transcripts, including downregulation in HbAS of several transcripts associated with severe malaria in collateral studies. Collectively, our comparative transcriptomic screen in vitro and in vivo indicates that P. falciparum adapts to HbAS by altering its protein chaperone and folding machinery, oxidative stress response, and protein export machinery. Because HbAS consistently protects from severe P. falciparum, modulation of these responses may offer avenues by which to neutralize P. falciparum parasites. IMPORTANCE Sickle-trait hemoglobin (HbAS) confers nearly complete protection from severe, life-threatening malaria, yet the molecular mechanisms that underlie HbAS protection from severe malaria remain incompletely understood. Here, we used transcriptome sequencing (RNA-seq) to measure the impact of HbAS on the blood-stage transcriptome of Plasmodium falciparum in in vitro time series experiments and in vivo samples from natural infections. Our in vitro time series data reveal that, during its blood stage, P. falciparum’s gene expression in HbAS is impacted primarily through alterations in the abundance of gene products as opposed to variations in the timing of gene expression. Collectively, our in vitro and in vivo data indicate that P. falciparum adapts to HbAS by altering its protein chaperone and folding machinery, oxidative stress response, and protein export machinery. Due to the persistent association of HbAS and protection from severe disease, these processes that are modified in HbAS may offer strategies to neutralize P. falciparum.

Sickle-trait hemoglobin (HbAS) confers near-complete protection from severe, life-threatening falciparum malaria in African children. Despite this clear protection, the molecular mechanisms by which HbAS confers these protective phenotypes remain incompletely understood. As a forward genetic screen for aberrant parasite transcriptional responses associated with parasite neutralization in HbAS red blood cells (RBCs), we performed comparative transcriptomic analyses ofPlasmodium falciparumin normal (HbAA) and HbAS erythrocytes during bothin vitrocultivation of reference parasite strains and naturally-occurringP. falciparuminfections in Malian children with HbAA or HbAS. Duringin vitrocultivation, parasites matured normally in HbAS RBCs, and the temporal expression was largely unperturbed of the highly ordered transcriptional program that underlies the parasite’s maturation throughout the intraerythrocytic development cycle (IDC). However, differential expression analysis identified hundreds of transcripts aberrantly expressed in HbAS, largely occurring late in the IDC. Surprisingly, transcripts encoding members of the Maurer’s clefts were overexpressed in HbAS despite impaired parasite protein export in these RBCs, while parasites in HbAS RBCs underexpressed transcripts associated with the endoplasmic reticulum and those encoding serine repeat antigen proteases that promote parasite egress. Analyses ofP. falciparumtranscriptomes from 32 children with uncomplicated malaria identified stage-specific differential expression: among infections composed of ring-stage parasites, only cyclophilin 19B was underexpressed in children with HbAS, while trophozoite-stage infections identified a range of differentially-expressed transcripts, including downregulation in HbAS of several transcripts associated with severe malaria in collateral studies. Collectively, our comparative transcriptomic screenin vitroandin vivoindicates thatP. falciparumadapts to HbAS by altering its protein chaperone and folding machinery, oxidative stress response, and protein export machinery. Because HbAS consistently protects from severeP. falciparum, modulation of these responses may offer avenues by which to neutralizeP. falciparumparasites.ImportanceSickle-trait hemoglobin (HbAS) confers near-complete protection from severe, life-threatening malaria, yet the molecular mechanisms that underlie HbAS protection from severe malaria remain incompletely understood. Here, we use RNA-seq to measure the impact of HbAS on the blood stage transcriptome ofPlasmodium falciparuminin vitrotime series experiments andin vivosamples from natural infections. Our in vitrotime series data reveal that, during its blood stage,P. falciparum’sgene expression in HbAS is impacted primarily through alterations in the abundance of gene products as opposed to variations in the timing of gene expression. Collectively, ourin vitroandin vivodata indicate thatP. falciparumadapts to HbAS by altering its protein chaperone and folding machinery, oxidative stress response, and protein export machinery. Due to the persistent association of HbAS and protection from severe disease, these processes that are modified in HbAS may offer strategies to neutralizeP. falciparum.

Background Drug resistance is one of the greatest challenges of malaria control programme in Mali. Recent advances in next-generation sequencing (NGS) technologies provide new and effective ways of tracking drug-resistant malaria parasites in Africa. The diversity and the prevalence of Plasmodium falciparum drug-resistance molecular markers were assessed in Dangassa and Nioro-du-Sahel in Mali, two sites with distinct malaria transmission patterns. Dangassa has an intense seasonal malaria transmission, whereas Nioro-du-Sahel has an unstable and short seasonal malaria transmission. Methods Up to 270 dried blood spot samples (214 in Dangassa and 56 in Nioro-du-Sahel) were collected from P. falciparum positive patients in 2016. Samples were analysed on the Agena MassARRAY® iPLEX platform. Specific codons were targeted in Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps, Pfarps10, Pfferredoxin, Pfexonuclease and Pfmdr2 genes. The Sanger’s 101-SNPs-barcode method was used to assess the genetic diversity of P. falciparum and to determine the parasite species. Results The Pfcrt_76T chloroquine-resistance genotype was found at a rate of 64.4% in Dangassa and 45.2% in Nioro-du-Sahel (p = 0.025). The Pfdhfr_51I-59R-108N pyrimethamine-resistance genotype was 14.1% and 19.6%, respectively in Dangassa and Nioro-du-Sahel. Mutations in the Pfdhps_S436-A437-K540-A581-613A sulfadoxine-resistance gene was significantly more prevalent in Dangassa as compared to Nioro-du-Sahel (p = 0.035). Up to 17.8% of the isolates from Dangassa vs 7% from Nioro-du-Sahel harboured at least two codon substitutions in this haplotype. The amodiaquine-resistance Pfmdr1_N86Y mutation was identified in only three samples (two in Dangassa and one in Nioro-du-Sahel). The lumefantrine-reduced susceptibility Pfmdr1_Y184F mutation was found in 39.9% and 48.2% of samples in Dangassa and Nioro-du-Sahel, respectively. One piperaquine-resistance Exo_E415G mutation was found in Dangassa, while no artemisinin resistance genetic-background were identified. A high P. falciparum diversity was observed, but no clear genetic aggregation was found at either study sites. Higher multiplicity of infection was observed in Dangassa with both COIL (p = 0.04) and Real McCOIL (p = 0.02) methods relative to Nioro-du-Sahel. Conclusions This study reveals high prevalence of chloroquine and pyrimethamine-resistance markers as well as high codon substitution rate in the sulfadoxine-resistance gene. High genetic diversity of P. falciparum was observed. These observations suggest that the use of artemisinins is relevant in both Dangassa and Nioro-du-Sahel.

Objectives To evaluate Plasmodium malariae susceptibility to current and lead candidate antimalarial drugs. Methods We conducted cross-sectional screening and detection of all Plasmodium species malaria cases, which were nested within a longitudinal prospective study, and an ex vivo assessment of efficacy of a panel of antimalarials against P. malariae and Plasmodium falciparum, both PCR-confirmed mono-infections. Reference compounds tested included chloroquine, lumefantrine, artemether and piperaquine, while candidate antimalarials included the imidazolopiperazine GNF179, a close analogue of KAF156, and the Plasmodium phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691. Results We report a high frequency (3%–15%) of P. malariae infections with a significant reduction in ex vivo susceptibility to chloroquine, lumefantrine and artemether, which are the current frontline drugs against P. malariae infections. Unlike these compounds, potent inhibition of P. malariae and P. falciparum was observed with piperaquine exposure. Furthermore, we evaluated advanced lead antimalarial compounds. In this regard, we identified strong inhibition of P. malariae using GNF179, a close analogue of KAF156 imidazolopiperazines, which is a novel class of antimalarial drug currently in clinical Phase IIb testing. Finally, in addition to GNF179, we demonstrated that the Plasmodium PI4K-specific inhibitor KDU691 is highly inhibitory against P. malariae and P. falciparum. Conclusions Our data indicated that chloroquine, lumefantrine and artemether may not be suitable for the treatment of P. malariae infections and the potential of piperaquine, as well as new antimalarials imidazolopiperazines and PI4K-specific inhibitor, for P. malariae cure.

Malaria transmission largely depends on environmental, climatic, and hydrological conditions. In Mali, malaria epidemiological patterns are nested within three ecological zones. This study aimed at assessing the relationship between those conditions and the incidence of malaria in Dangassa and Koila, Mali. Malaria data was collected through passive case detection at community health facilities of each study site from June 2015 to January 2017. Climate and environmental data were obtained over the same time period from the Goddard Earth Sciences (Giovanni) platform and hydrological data from Mali hydraulic services. A generalized additive model was used to determine the lagged time between each principal component analysis derived component and the incidence of malaria cases, and also used to analyze the relationship between malaria and the lagged components in a multivariate approach. Malaria transmission patterns were bimodal at both sites, but peak and lull periods were longer lasting for Koila study site. Temperatures were associated with malaria incidence in both sites. In Dangassa, the wind speed (p = 0.005) and river heights (p = 0.010) contributed to increasing malaria incidence, in contrast to Koila, where it was humidity (p &lt, 0.001) and vegetation (p = 0.004). The relationships between environmental factors and malaria incidence differed between the two settings, implying different malaria dynamics and adjustments in the conception and plan of interventions.

Background Seasonal malaria chemoprevention (SMC) is a new strategy to prevent malaria in children under 5 years old. It has been recommended by the World Health Organization since 2012 in malaria-endemic areas with seasonal transmission. This study aimed to assess the changes in malaria indicators through two consecutive years of SMC routine implementation in children under 5 years old in Dangassa, where malaria is endemic with a long and high transmission season. Methods From 2012 to 2016, a cohort study was conducted in Dangassa village. The study team based in the village followed all malaria clinical cases in children under 5 years old at the community health centre. During the study, SMC was routinely implemented in collaboration with the National Malaria Control Programme. The Cox regression model was used in order to compare malaria risk during the study. Results The Cox regression model showed a significant reduction in malaria clinical incidence, both in 2015 (HR = 0.27 (0.18–0.40), 95% CI) and in 2016 (HR = 0.23 (0.15–0.35), 95% CI) of SMC implementation compared to October 2013. Gametocyte and fever prevalence was lower between September and October during SMC implementation (2015 and 2016) compared to the same period before SMC implementation (2013–2014). A slight increase of malaria incidence was observed in December at the end of SMC implementation. Conclusion SMC has significantly reduced both malaria incidence and gametocyte prevalence and improved haemoglobin levels in children under 5 years old after 2 years of routine implementation.

Standard hematological indices are commonly used in malaria epidemiological studies to measure anemia prevalence and calculate blood parasite densities. In Africa, few studies have investigated how these indices change during a malaria transmission season and with increasing age. To address these knowledge gaps, we collected blood from 169 healthy Malian children aged 3-12 years before (May 2010) and after (January 2011) a transmission season. Red blood cell (RBC) count, hemoglobin (Hb) level, hematocrit (Ht), white blood cell (WBC) count, and WBC subsets were measured in paired blood samples, and the data were stratified by month (May, January) and age group (3-5, 6-8, and 9-12 years). From May to January, RBC count (4.53-4.70 × 10(6)/μL; P < 0.0001), Hb level (11.5-11.9 g/dL; P < 0.0001), and Ht (37.1-39.2%; P < 0.0001) increased, and WBC count (6.46-5.96 × 10(3)/μL; P = 0.0006) decreased. From May to January, the prevalence of WBC subsets also changed: 35-43% neutrophils, 6.5-7.6% monocytes, and 53-45% lymphocytes (P < 0.001). These seasonal changes were not associated with the number of malaria episodes experienced in the interim or the presence of RBC polymorphisms. In May, Hb (11.2, 11.4, and 11.8 g/dL; P = 0.0013) and Ht (36.5%, 36.7%, and 38.1%; P = 0.0154) increased and WBC count (8.04, 6.43, and 5.76 × 10(3)/μL; P < 0.0001) decreased with age group; similar differences were observed in January. These data suggest that season- and age-based reference values for hematological indices are needed to better estimate anemia prevalence and parasite density in malaria epidemiological studies.

Erythrocyte G6PD deficiency is the most common worldwide enzymopathy. The aim of this study was to determine erythrocyte G6PD deficiency in 3 ethnic groups of Mali and to investigate whether erythrocyte G6PD deficiency was associated to the observed protection against malaria seen in Fulani ethnic group. The study was conducted in two different areas of Mali: in the Sahel region of Mopti where Fulani and Dogon live as sympatric ethnic groups and in the Sudanese savannah area where lives mostly the Malinke ethnic group. The study was conducted in 2007 in Koro and in 2008 in Naguilabougou. It included a total 90 Dogon, 42 Fulani and 80 Malinke ethnic groups. Malaria was diagnosed using microscopic examination after Giemsa-staining of thick and thin blood smear. G6PD deficiency (A-(376/202)) samples were identified using RFLP (Restriction Fragment Length Polymorphism) assay and analysis of PCR-amplified DNA amplicon. G6PD deficiency (A-(376/202)) rate was 11.1%, 2.4%, and 13.3% in Dogon, Fulani, and Malinke ethnic group respectively. Heterozygous state for G6PD (A-(376/202)) was found in 7.8% in Dogon; 2.4% in Fulani and 9.3% in Malinke ethnic groups while hemizygous state was found at the frequency of 2.2% in Dogon and 4% in Malinke. No homozygous state was found in our study population.We conclude that G6PD deficiency is not differing significantly between the three ethnic groups, Fulani, Dogon and Malinke.

Artemisinins rapidly kill all asexual blood stages of Plasmodium falciparum [1], including young ring forms before they sequester in the microvessels of vital organs, and are thus more effective than quinolines in reducing malaria morbidity and mortality [2, 3]. For this reason, artemisinin-based combination therapies (ACTs) are first-line treatments for P. falciparum malaria worldwide [4]. ACTs are the use of artemisinin or one of its derivatives—artesunate, artemether, dihydroartemisinin (DHA)—in combination with a partner drug. DHA, the active metabolite of all artemisinins, has a short half-life (approximately 1–3 hours) in plasma; therefore, partner drugs with longer half-lives (ie, approximately 7–21 days) are needed to eliminate residual parasites [5, 6]. Parasite resistance to nearly every commonly used partner drug is entrenched or emerging in western Cambodia [7]. Reports of artemisinin resistance in western Cambodia [8, 9] and western Thailand [10]—where antimalarial-resistant parasites have previously emerged and spread to Africa [11–13]—are thus extremely worrisome. This phenotype manifests as a slow parasite clearance rate in response to an artemisinin taken orally to treat uncomplicated malaria [14]. Unlike quinoline resistance, artemisinin resistance does not associate with known molecular markers of drug resistance or reduced parasite drug susceptibility in vitro [8, 9, 15]. Slow parasite clearance may be partly defined as a parasite-heritable trait [9, 10, 16] and was recently associated with a major region of the parasite genome [17]. Microsatellite-defined parasite genetics, however, has not been able to account for all the variation in parasite clearance rates in Cambodia and Thailand [9, 10, 16]. Few studies have explored how host factors, such as naturally acquired immunity and red blood cell (RBC) polymorphisms, influence parasite clearance rates in response to artemisinins. A retrospective analysis of 18 699 patients with P. falciparum malaria treated with artemisinins found that parasite clearance was slowest in low-transmission settings and relatively fast in high-transmission settings, suggesting that acquired immunity accelerates parasite clearance [18]. A recent prospective study suggested that hemoglobin (Hb) E is associated with slow parasite clearance rate in Pursat, western Cambodia [9], but the effect of acquired immunity on this parameter is not well defined. This is because, in southeast Asia, age is an inadequate surrogate of immunity to malaria and no in vitro correlate of parasite-clearing immunity has been identified. Few parasite clearance rates have been reported from Africa [19], where ACTs were recently introduced and artemisinin monotherapies have been used for 15 years in some areas [20]. The proposed mechanism of action of artemisinins involves endoperoxide-derived, free radicals that alkylate and oxidize the proteins and lipids of intraerythrocytic parasites [21]. Artemisinin-treated parasites undergo pyknosis rapidly in vivo and are cleared from the bloodstream by “pitting” in the spleen, which returns previously infected, intact RBCs back into circulation (Figure ​(Figure11A) [22–25]. Although largely determined by the rates of pyknosis and pitting, the parasite clearance rate is nonetheless a complex phenotype produced by a combination of parasite and host factors. Parasite expression of cytoadherence ligands, for example, can determine when late ring-stage parasites sequester in microvessels and disappear from peripheral blood (Figure ​(Figure11C) [26]. If parasites sequester en masse [26] while parasite clearance is being monitored, parasite densities may decrease markedly by an artemisinin-independent mechanism. Host antibody responses that prevent sequestration and opsonize parasitized RBCs may also accelerate parasite clearance (Figure ​(Figure11B). Figure 1. Clearance of ring-stage Plasmodium falciparum parasites from peripheral blood during a parasite clearance rate study. Dihydroartemisinin, the active metabolite of all artemisinins, causes ring-stage parasites to undergo pyknosis (A). These circulating ... To obtain baseline surveillance data for the emergence or spread of artemisinin resistance in Africa and to investigate the role of acquired immunity in parasite clearance, we measured parasite clearance rates in response to artesunate in a high transmission area of Mali, where ACTs were introduced only 2 years previously and where children rapidly acquire antimalarial immunity with age.

In 2006, the Malian government established a program for free insecticide-treated net (ITNs) distribution during antenatal care visit (ANC) and intermittent preventive treatment during pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) for pregnant women. In March to November of 2009, we conducted a cross-sectional study in peri-urban areas of Bamako, Mali to determine the malaria prevalence among pregnant women and their newborn children in the context of this policy. We included 379 pregnant women aged 15 to 45 years. At delivery, malaria was diagnosed using peripheral thick smears in mothers and newborns, as well as umbilical cord blood and placental blood. The prevalence of Plasmodium falciparum malaria was 2.4, 1.6 and 0.5% respectively in mother, placenta and cord samples; we observed a low birth weight rate of 12.1%. Approximately 77% of our parturient were housewives. The illiteracy rate among this group was 72.3%. Of the 379 women, 73% had at least three prenatal visits, 83% had received at least one free ITNs and 72% had received IPTp-SP during antenatal visit. Among them, 81% claimed to have complied with IPTp-SP. No congenital malaria was found. The prevalence of malaria in both mother and newborn has show a significant decrease in Bamako, compared with previous studies before the implementation of IPTp-SP policy in Mali. A high rate of coverage and use of IPTp-SP and ITNs correlate with lower malaria prevalence in pregnant women.

International audience; During their lifespan, circulating RBC are frequently checked for their deformability. This mechanical quality control operates essentially in the human spleen. RBC unable to squeeze though narrow splenic slits are retained and cleared from the blood circulation. Under physiological conditions this prevents microvessels from being clogged by senescent, rigid RBC. Retention of poorly deformable RBC is an important determinant of pathogenesis in malaria and may also impact the clinical benefit of transfusion. Modulating the splenic retention of RBC has already been proposed to support therapeutic approaches in these research fields. To this aim, the development of microplates for high throughput filtration of RBC through microsphere layers (microplate-based microsphiltration) has been undertaken. This review focuses on potential therapeutic applications provided by this technology in malaria chemotherapy and transfusion.

Sickle hemoglobin (Hb) S and HbC may protect against malaria by reducing the expression of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of parasitized red blood cells (RBCs), thereby weakening their cytoadherence to microvascular endothelial cells (MVECs) and impairing their activation of MVECs to produce pathological responses. Therefore, we hypothesized that parasites causing malaria in HbAS or HbAC heterozygotes have overcome this protective mechanism by expressing PfEMP1 variants which mediate relatively strong binding to MVECs. To test this hypothesis, we performed 31 cytoadherence comparisons between parasites from HbAA and HbAS (or HbAC) Malian children with malaria. Ring-stage parasites from HbAA and HbAS (or HbAC) children were cultivated to trophozoites, purified, and then inoculated in parallel into the same wildtype uninfected RBCs. After one cycle of invasion and maturation to the trophozoite stage expressing PfEMP1, parasite strains were compared for binding to MVECs. In this assay, there were no significant differences in the binding of parasites from HbAS and HbAC children to MVECs compared to those from HbAA children (HbAS, fold-change = 1.46, 95% CI 0.97-2.19, p = 0.07; HbAC, fold-change = 1.19, 95% CI 0.77-1.84, p = 0.43). These data suggest that in-vitro reductions in cytoadherence by HbS and HbC may not be selecting for expression of high-avidity PfEMP1 variants in vivo. Future studies that identify PfEMP1 domains or amino-acid motifs which are selectively expressed in parasites from HbAS children may provide further insights into the mechanism of malaria protection by the sickle-cell trait.

Background Plasmodium falciparum elicits host inflammatory responses that cause the symptoms and severe manifestations of malaria. One proposed mechanism involves formation of immunostimulatory uric acid (UA) precipitates, which are released from sequestered schizonts into microvessels. Another involves hypoxanthine and xanthine, which accumulate in parasitized red blood cells (RBCs) and may be converted by plasma xanthine oxidase to UA at schizont rupture. These two forms of ‘parasite-derived’ UA stimulate immune cells to produce inflammatory cytokines in vitro. Methods and Findings We measured plasma levels of soluble UA and inflammatory cytokines and chemokines (IL-6, IL-10, sTNFRII, MCP-1, IL-8, TNFα, IP-10, IFNγ, GM-CSF, IL-1β) in 470 Malian children presenting with uncomplicated malaria (UM), non-cerebral severe malaria (NCSM) or cerebral malaria (CM). UA levels were elevated in children with NCSM (median 5.74 mg/dl, 1.21-fold increase, 95% CI 1.09–1.35, n = 23, p = 0.0007) and CM (median 5.69 mg/dl, 1.19-fold increase, 95% CI 0.97–1.41, n = 9, p = 0.0890) compared to those with UM (median 4.60 mg/dl, n = 438). In children with UM, parasite density and plasma creatinine levels correlated with UA levels. These UA levels correlated with the levels of seven cytokines [IL-6 (r = 0.259, p

Background Wuchereria bancrofti (Wb) and Mansonella perstans (Mp) are blood-borne filarial parasites that are endemic in many countries of Africa, including Mali. The geographic distribution of Wb and Mp overlaps considerably with that of malaria, and coinfection is common. Although chronic filarial infection has been shown to alter immune responses to malaria parasites, its effect on clinical and immunologic responses in acute malaria is unknown. Methodology/Principal Findings To address this question, 31 filaria-positive (FIL+) and 31 filaria-negative (FIL−) children and young adults, matched for age, gender and hemoglobin type, were followed prospectively through a malaria transmission season. Filarial infection was defined by the presence of Wb or Mp microfilariae on calibrated thick smears performed between 10 pm and 2 am and/or by the presence of circulating filarial antigen in serum. Clinical malaria was defined as axillary temperature ≥37.5°C or another symptom or sign compatible with malaria infection plus the presence of asexual malaria parasites on a thick blood smear. Although the incidence of clinical malaria, time to first episode, clinical signs and symptoms, and malaria parasitemia were comparable between the two groups, geometric mean hemoglobin levels were significantly decreased in FIL− subjects at the height of the transmission season compared to FIL+ subjects (11.4 g/dL vs. 12.5 g/dL, p, Author Summary In many regions of the world, including sub-Saharan Africa, concomitant infection with multiple parasites is common. In order to examine the effects of filariasis, a chronic helminth infection, on immune responses and clinical manifestations of acute malaria infection, the authors followed 31 filaria-infected (FIL+) and 31 filaria-uninfected (FIL–) individuals living in a malaria-endemic area of Mali through an entire malaria transmission season for the development of clinical malaria (fever or other symptoms of malaria in the setting of detectable blood parasites). Serum levels of inflammatory cytokines previously associated with severe malaria were decreased in FIL+ subjects at the time of acute clinical malaria. Although there were no differences between FIL+ and FIL– subjects with respect to the time of first episode of malaria or the number or severity of malaria episodes, filarial infection appeared to protect against the development of anemia during the malaria transmission season. These findings demonstrate that chronic filarial infection modulates the immune response to acute malaria. The apparent effect on anemia is intriguing and deserves further study.

Background Artemisinin-based combination therapy (ACT) is currently the most effective medicine for the treatment of uncomplicated malaria. Artemisinin has previously been shown to increase the clearance of Plasmodium falciparum in malaria patients with haemoglobin E trait, but it did not increase parasite inhibition in an in vitro study using haemoglobin AS erythrocytes. The current study describes the efficacy of artemisinin derivatives on P. falciparum clearance in patients with glucose-6-phosphate dehydrogenase deficiency (G6PD), a haemoglobin enzyme deficiency, not yet studied in the same context, but nonetheless is a common in malaria endemic areas, associated with host protection against uncomplicated and severe malaria. The impact of G6PD deficiency on parasite clearance with ACT treatment was compared between G6PD-deficient patients and G6PD-normal group. Methods Blood samples from children and adults participants (1 to 70 years old) with uncomplicated P. falciparum malaria residing in Kambila, Mali were analysed. Study participants were randomly assigned to receive either artemether-lumefantrine (Coartem®) or artesunate plus mefloquine (Artequin™). A restriction-fragment length polymorphism analysis of PCR-amplified DNA samples was used to identify the (A-) allele of the gene mutation responsible for G6PD deficiency (G6PD*A-). 470 blood samples were thus analysed and of these, DNA was extracted from 315 samples using the QIAamp kit for PCR to identify the G6PD*A- gene. Results The DNA amplified from 315 samples using PCR showed that G6PD*A- deficiency was present in 56 participants (17.8%). The distribution of the specific deficiency was 1%, 7% and, 9.8% respectively for homozygous, hemizygous, and heterozygous genotypes. Before treatment, the median parasitaemia and other baseline characteristics (mean haemoglobin, sex and age groups) between G6PD deficiency (hemizygous, heterozygous, and homozygous) and G6PD-normal participants were comparable (p > 0.05). After treatment, parasite clearance did not change significantly whether the participants were G6PD deficient or G6PD normal on day 1 (OR = 1.3; CI = 0.70-2.47; p > 0.05) and on day 2 (OR = 0.859; CI = 0.097-7.61; p > 0.05). Conclusions The presence of G6PD deficiency does not appear to significantly influence the clearance of P. falciparum in the treatment of uncomplicated malaria using ACT.

The World Health Organization (WHO) recently recommended that the time to first malaria episode serve as the primary end point in phase III malaria vaccine trials--the first of which will be held in Africa. Although common red blood cell (RBC) polymorphisms such as sickle hemoglobin (HbS) are known to protect against malaria in Africa, their impact on this end point has not been investigated.A longitudinal study of 225 individuals aged 2-25 years was conducted in Mali. The association between common RBC polymorphisms and the time to first malaria episode was evaluated.Among children aged 2-10 years, sickle cell trait (HbAS) was associated with a 34-day delay in the median time to first malaria episode (P= .017) Cox regression analysis showed that greater age (hazard ratio [HR], 0.87 [95% CI, 0.80-0.94]; (P= .001), HbAS (HR, 0.48 [95% CI, 0.26-0.91]; (P= .024), and asymptomatic parasitemia at enrollment (HR, 0.35 [95% CI, 0.14-0.85]; (P= .021) were associated with decreased malaria risk.Given the delay in the time to first malaria episode associated with HbAS, it would be advisable for clinical trials and observational studies that use this end point to include Hb typing in the design of studies conducted in areas where HbAS is prevalent.

Sickle trait, the heterozygous state of normal hemoglobin A (HbA) and sickle hemoglobin S (HbS), confers protection against malaria in Africa. AS children infected with Plasmodium falciparum are less likely than AA children to suffer the symptoms or severe manifestations of malaria, and they often carry lower parasite densities than AA children. The mechanisms by which sickle trait might confer such malaria protection remain unclear. We have compared the cytoadherence properties of parasitized AS and AA erythrocytes, because it is by these properties that parasitized erythrocytes can sequester in postcapillary microvessels of critical tissues such as the brain and cause the life-threatening complications of malaria. Our results show that the binding of parasitized AS erythrocytes to microvascular endothelial cells and blood monocytes is significantly reduced relative to the binding of parasitized AA erythrocytes. Reduced binding correlates with the altered display of P. falciparum erythrocyte membrane protein-1 (PfEMP-1), the parasite's major cytoadherence ligand and virulence factor on the erythrocyte surface. These findings identify a mechanism of protection for HbS that has features in common with that of hemoglobin C (HbC). Coinherited hemoglobin polymorphisms and naturally acquired antibodies to PfEMP-1 may influence the degree of malaria protection in AS children by further weakening cytoadherence interactions.

SCT is a heterozygous genetic condition resulting from a single point mutation in the gene for the beta chain of hemoglobin (hemoglobin S, HbS), by which an encoded glutamate at position six is replaced with valine [1,2]. The prevalence of hemoglobin S in regions where it provides protection against malaria is a textbook example of balanced polymorphism and natural selection. G6PD is an enzyme which catalyzes the first step of the pentose phosphate pathway and supports the production of reduced glutathione, a major defense against oxidant stress in red blood cells. G6PD deficiency is an X-linked condition associated with damage to red blood cells and hemolysis in response to a variety of exposures, including infection, ingestion of fava beans, or exposure to certain chemicals or medications [3,4]. A number of epidemiological studies have associated SCT or G6PD deficiency with protection against malaria, in particular severe, life-threatening malaria in its cerebral form [5–9]. Protection by SCT and G6PD deficiency are usually thought to act independently, but no studies have reported whether the protective effects of these two conditions are synergistic, are neutrally additive, or interfere with one another. Here we report on the occurrence of uncomplicated and severe malaria in Malian children with SCT, the G6PD (A−) polymorphism, or both of these conditions together. Among children with SCT, the prevalence of severe versus uncomplicated malaria in children with the G6PD (A−) polymorphism was 12.2%. When evaluated by sex, this prevalence was 6.6% and 28.5% in hemizygous males and heterozygous females, respectively. These results raise the possibility that the heterozygous G6PD (A−) condition and sickle trait do not supplement and may even interfere with one another in their protection against severe malaria (OR = 15; 95CI 2.07–132.31; P = 0.003). SCT and G6PD (A−) deficiency are frequently cited examples of red blood cell polymorphisms that confer resistance to malaria. Despite the different hemoglobin and enzyme functions that are affected, these polymorphisms are prevalent at comparably high frequencies in many populations of Mali and other regions of Africa [10,11]. These distributions are generally thought to reflect natural selection from the protections that SCT and G6PD (A−) deficiency independently provide against severe malaria [12,13]. Results of the work presented here confirm the association of SCT with protection against severe malaria (P = 0.01). This result corroborates with that of Doumbo et al. (1992) who reported on severe malaria children in a pediatric hospital of Mali. Another study likewise reported a protective effect of SCT against severe forms of malaria in the neighboring country of Burkina Faso [14]. Curiously, our study found that SCT provides significant protection against severe malaria in female but not male children of the villages of Kangaba and Kela (Table I). The reason for this differential signal of protection in the children is not known. Whether it may relate to additional sex-linked genetic factors or particular environmental conditions of the villages is a question that will require further investigation. Table I Distribution of Hemoglobin Types Among Uncomplicated and Severe Cases Malaria in Malian Children In a previous article, we reported that the hemizygous G6PD (A−) condition in the male children of Kangaba and Kela is associated with protection against severe malaria [9]. With this evidence in hand and also with evidence for a high degree of severe protection by SCT in female children, we tested whether an enhanced protective effect might be detected when the G6PD (A−) polymorphism and SCT were present in combination. In hemoglobin AS females heterozygous for G6PD (A−), the occurrence of severe malaria in four individuals suggested no synergistic or additive effect of the two conditions; instead, an OR of 15 (95%CI 2.07–132.31; P = 0.003) suggested the possibility of interference (negative epistasis) between G6PD (A−) heterozygosity and sickle trait in the female children (Table II). However, in sickle trait males with the hemizygous G6PD (A−) condition, no such evidence was found negative epistasis between sickle trait and G6PD (A−) heterozygosity (OR = 0.47; 95CI 0.07–2.40*; P = 0.53). This may reflect no interference due to an absence of a detectable protective activity from SCT in the male children of Kangaba and Kela (discussed above). However, considering the low numbers of severe malaria cases with both sickle trait and a G6PD (A−) condition in our study, additional studies with greater numbers of subjects will be required to further evaluate the possibilities suggested here. Table II Prevalence of G6PD Types in Malian Sickle Trait Children with Uncomplicated or Severe Malaria Our observations invite consideration of the molecular mechanisms by which various erythrocyte polymorphisms protect against severe malaria. A report from Kenya recently provided evidence for negative epistasis between SCT and alpha-thalassemia in protection against malaria [16]. How the protective mechanisms of these and other conditions including G6PD (A−) might interact to mutually interfere in malaria protection remains to be resolved. By the way, more recent study with a limited number of patients has shown that G6PD deficiency and SCT have no effect on the severity of anemia in children infected with both HIV-1 and P. falciparum [17] despite the high proportion of G6PD deficiency in coinfected children. Further study with large sample size is needed to confirm this observation. Understanding the interaction of malaria protective factors in various infectious diseases may be a valuable tool of medical interest. Our evaluation of data was previously collected in the villages of Kangaba and Kela, Mali [9]. Subjects in the case–control study included children 10 years or less of age who presented with clinical symptoms of malaria. Microscopic confirmations of parasitemia, assignments of the cases into uncomplicated or severe malaria, and hemoglobin typing and G6PD (A−) detection were as described [9]. Data evaluation and analysis were performed under consents and protocols approved by Institutional Review Boards of the National Institute of Allergy and Infectious Diseases, Bethesda, MD and of the University of Bamako, Mali.

Background α-thalassemia results from decreased production of α-globin chains that make up part of hemoglobin tetramers (Hb; α2β2) and affects up to 50% of individuals in some regions of sub-Saharan Africa. Heterozygous (−α/αα) and homozygous (−α/−α) genotypes are associated with reduced risk of severe Plasmodium falciparum malaria, but the mechanism of this protection remains obscure. We hypothesized that α-thalassemia impairs the adherence of parasitized red blood cells (RBCs) to microvascular endothelial cells (MVECs) and monocytes – two interactions that are centrally involved in the pathogenesis of severe disease. Methods and Findings We obtained P. falciparum isolates directly from Malian children with malaria and used them to infect αα/αα (normal), −α/αα and −α/−α RBCs. We also used laboratory-adapted P. falciparum clones to infect −/−α RBCs obtained from patients with HbH disease. Following a single cycle of parasite invasion and maturation to the trophozoite stage, we tested the ability of parasitized RBCs to bind MVECs and monocytes. Compared to parasitized αα/αα RBCs, we found that parasitized −α/αα, −α/−α and −/−α RBCs showed, respectively, 22%, 43% and 63% reductions in binding to MVECs and 13%, 33% and 63% reductions in binding to monocytes. α-thalassemia was associated with abnormal display of P. falciparum erythrocyte membrane protein 1 (PfEMP1), the parasite’s main cytoadherence ligand and virulence factor, on the surface of parasitized RBCs. Conclusions Parasitized α-thalassemic RBCs show PfEMP1 display abnormalities that are reminiscent of those on the surface of parasitized sickle HbS and HbC RBCs. Our data suggest a model of malaria protection in which α-thalassemia ameliorates the pro-inflammatory effects of cytoadherence. Our findings also raise the possibility that other unstable hemoglobins such as HbE and unpaired α-globin chains (in the case of β-thalassemia) protect against life-threatening malaria by a similar mechanism.

Background Sickle-cell trait (HbAS) reduces falciparum malaria risk and suppresses parasitaemia. Although several candidate mechanisms have been proposed, their epidemiological, clinical and experimental correlates have not been adequately explained. To explore the basis for generally lower parasitaemias and delayed malaria episodes in children with HbAS, it is hypothesized here that their spleen-dependent removal of ring-infected red blood cells (RBCs) is more efficient than in children with normal haemoglobin A (HbAA). Methods The mechanical splenic retention of Plasmodium falciparum-infected RBCs from subjects with HbAS or HbAA was investigated using two physiologically relevant methods: microsphiltration and ex vivo spleen perfusion. P. falciparum-infected RBCs obtained from in vitro cultures and from patients were used in either normoxic or hypoxic conditions. The effect of sickling in ring-infected HbAS RBCs was also investigated. Results When a laboratory-adapted parasite strain was analysed, ring-infected HbAA RBCs were retained in microsphilters at similar or greater levels than ring-infected HbAS RBCs, under normoxic (retention rate 62.5 vs 43.8 %, P