Understanding Human Cerebral Malaria through a Blood Transcriptomic Signature: Evidences for Erythrocyte Alteration, Immune/Inflammatory Dysregulation, and Brain Dysfunction.

BACKGROUND: Cerebral malaria (CM), a reversible encephalopathy affecting young children, is a medical emergency requiring rapid clinical assessment and treatment. However, understanding of the genes/proteins and the biological pathways involved in the disease outcome is still limited. METHODS: We have performed a whole transcriptomic analysis of blood samples from Malian children with CM or uncomplicated malaria (UM). Hierarchical clustering and pathway, network, and upstream regulator analyses were performed to explore differentially expressed genes (DEGs). We validated gene expression for 8 genes using real-time quantitative PCR (RT-qPCR). Plasma levels were measured for IP-10/CXCL10 and IL-18. RESULTS: A blood RNA signature including 538 DEGs (∣FC | ≥2.0, adjusted P value ≤ 0.01) allowed to discriminate between CM and UM. Ingenuity Pathway Analysis (IPA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed novel genes and biological pathways related to immune/inflammatory responses, erythrocyte alteration, and neurodegenerative disorders. Gene expressions of CXCL10, IL12RB2, IL18BP, IL2RA, AXIN2, and NET were significantly lower in CM whereas ARG1 and SLC6A9 were higher in CM compared to UM. Plasma protein levels of IP-10/CXCL10 were significantly lower in CM than in UM while levels of IL-18 were higher. Interestingly, among children with CM, those who died from a complication of malaria tended to have higher concentrations of IP-10/CXCL10 and IFN-γ than those who recovered. CONCLUSIONS: This study identified some new factors and mechanisms that play crucial roles in CM and characterized their respective biological pathways as well as some upstream regulators.

Gene Expression Analysis Reveals Genes Common to Cerebral Malaria and Neurodegenerative Disorders.

Cerebral malaria, a reversible encephalopathy affecting young children, is a medical emergency requiring urgent clinical assessment and treatment. We performed a whole-transcriptomic analysis of blood samples from Malian children with cerebral or uncomplicated malaria. We focused on transcripts from pathways for which dysfunction has been associated with neurodegenerative disorders. We found that SNCA, SIAH2, UBB, HSPA1A, TUBB2A, and PINK1 were upregulated (fold-increases, ≥2.6), whereas UBD and PSMC5 were downregulated (fold-decreases, ≤4.39) in children with cerebral malaria, compared with those with uncomplicated malaria. These findings provide the first evidence for pathogenic mechanisms common to human cerebral malaria and neurodegenerative disorders.

The IL17F and IL17RA Genetic Variants Increase Risk of Cerebral Malaria in Two African Populations.

Cerebral malaria (CM) is a neurological complication of infection with Plasmodium falciparum that is partly caused by cytokine-mediated inflammation. It is not known whether interleukin-17 (IL-17) cytokines, which regulate inflammation, control the development of CM. To evaluate the involvement of IL-17 cytokines in CM, we analyzed 46 common polymorphisms in IL17A, IL17F, and IL17RA (which encodes the common receptor chain of the members of the IL-17 family) in two independent African populations. A case-control study involving 115 Nigerian children with CM and 160 controls from the community (CC) showed that IL17F reference single nucleotide polymorphism (SNP) 6913472 (rs6913472) (P = 0.004; odds ratio [OR] = 3.12), IL17F rs4715291 (P = 0.004; OR = 2.82), IL17RA rs12159217 (P = 0.01; OR = 2.27), and IL17RA rs41396547 (P = 0.026; OR = 3.15) were independently associated with CM. A replication study was performed in 240 nuclear Malian family trios (two parents with one CM child). We replicated the association for 3 SNPs, IL17F rs6913472 (P = 0.03; OR = 1.39), IL17RA rs12159217 (P = 0.01; OR = 1.52), and IL17RA rs41396547 (P = 0.04; OR = 3.50). We also found that one additional SNP, IL17RA rs41433045, in linkage disequilibrium (LD) with rs41396547, was associated with CM in both Nigeria and Mali (P = 0.002; OR = 4.12 in the combined sample). We excluded the possibility that SNPs outside IL17F and IL17RA, in strong LD with the associated SNPs, could account for the observed associations. Furthermore, the results of a functional study indicated that the aggravating GA genotype of IL17F rs6913472 was associated with lower IL-17F concentrations. Our findings show for the first time that IL17F and IL17RA polymorphisms modulate susceptibility to CM and provide evidence that IL-17F protects against CM.

Genotype combinations of two IL4 polymorphisms influencing IL-4 plasma levels are associated with different risks of severe malaria in the Malian population.

We have previously found that children heterozygous for IL4 variable-number tandem repeat (VNTR) (rs8179190) or IL4-33 (rs2070874) variants were at risk for severe malaria (SM), whereas homozygous children were protected suggesting a complex genetic control. Hence, to dissect this complex genetic control of IL4 VNTR and IL4-33, we performed further investigation by conditional logistic regression analysis and found a strong interaction between both markers (p < 10(-6)). The best-fit model revealed three genotype combinations associated with different levels of SM risk. The highest risk (odds ratio (OR) = 4.8, 95% confidence interval (CI) = 2.0-11.5) was observed for subjects carrying at least one copy of both IL4-33 allele T and IL4 VNTR allele 1, who exhibited higher interleukin (IL)-4 plasma levels (p = 0.007). Children homozygous for IL4 VNTR allele 2 had a lower SM risk as well as lower IL-4 plasma levels. Our findings indicate that the genetic interaction between these two IL-4 variants is a key factor of SM susceptibility, probably because of its direct role in IL-4 regulation.

Molecular incidence and clearance of Plasmodium falciparum infection.

BACKGROUND: Although the epidemiology of malaria has been based primarily on microscopy and rapid diagnostic tests, molecular methods are necessary to understand the complexity of natural infection in regions where transmission is intense and simultaneous infection with multiple parasite genotypes is common such as sub-Saharan Africa. METHODS: To compare microscopic and molecular estimates of the incidence and clearance of Plasmodium falciparum infection, we followed 80 children monthly for 1 year in the village of Bancoumana in Mali. RESULTS AND DISCUSSION: Similar seasonal patterns were observed with both methods (rainy season peak, dry season nadir), although molecular methods detected more infections than microscopy (571 vs 331 in 906 specimens), more new infections (311 vs 104 during 829 person-months) and spontaneous clearance events (317 vs 116) and found higher incidence (0.38 vs 0.13 new genotypes/person/month, p < 0.001) and spontaneous clearance rates (0.38 vs 0.14 genotypes cleared/person/month, p < 0.001). These differences were greatest for persistently-infected subjects in whom neither new infections nor the clearance of old infections could be detected by microscopy (0.71 new infections and 0.73 cleared infections per month using molecular methods vs 0.000 by microscopy, p < 0.001). CONCLUSIONS: Molecular methods provide information about genetic diversity, the intensity of transmission and spontaneous clearance in the absence of drug treatment that cannot be obtained by microscopy. They will be necessary to evaluate the efficacy of vaccines, drugs and other control strategies for diseases such as malaria in which simultaneous infection with more than one organism (genotype) is common.

Inter-observer agreement according to malaria parasite density.

BACKGROUND: Recent developments in diagnostic techniques for malaria, particularly DNA probes and sero-immunology, have raised questions as to how these techniques might be used to facilitate malaria diagnosis at the most peripheral levels of the primary health care system. At present, malaria diagnosis is based on the standard microscopic examination of blood films in most field epidemiologic studies and is likely to remain so in the immediate future in Africa. The objective of this study was to assess inter-observer agreement for the examination of Giemsa-stained slides for Plasmodium falciparum parasites. METHODS: Children aged 0 to 10 years were enrolled yearly in Bancoumana village (West Africa), mainly during the transmission season (June to October). The blood smears obtained from the persistently negative children in June 1996, August 1996, October 1996 and March 1997 were systematically re-examined. A stratified random sample (10%) proportional to the following parasite density classes 1-100, 101-5000, and 5001 and over was taken from the slides collected. The kappa statistics and the intra-class correlation were used as measures of agreement the first and the second slide examinations. RESULTS: The weighted kappa statistic, widely used as a chance-corrected measure for nominal agreement, showed excellent inter-observer agreement (κ(w)=0.7926; 95% CI [0.7588, 0.8263]; p=0.01). The intra-class correlation co-efficient had the same value of 0.7926 confirming the appropriateness of the weighted kappa statistic. Inter-observer agreement for slides read as negative by one observer, or as containing more than 100 parasites per µl, was excellent: 97% (493/506) and 92% (145/158), respectively. In contrast, the inter-observer agreement for slides read by one observer as containing 1-100 parasites/µl was poor, 36% (96/268). CONCLUSIONS: In field conditions in Mali, there was a high reproducibility for slides reported as negative or as having more than 100 parasites per µl. However, smears with readings of 1-100 parasites per µl were less reproducible and should be re-examined carefully.

Sickle cell trait protects against Plasmodium falciparum infection.

Although sickle cell trait protects against severe disease due to Plasmodium falciparum, it has not been clear whether sickle trait also protects against asymptomatic infection (parasitemia). To address this question, the authors identified 171 persistently smear-negative children and 450 asymptomatic persistently smear-positive children in Bancoumana, Mali (June 1996 to June 1998). They then followed both groups for 2 years using a cohort-based strategy. Among the 171 children with persistently negative smears, the median time for conversion to smear-positive was longer for children with sickle trait than for children without (274 vs. 108 days, P < 0.001; Cox hazard ratio = 0.56, 95% confidence interval: 0.33, 0.96; P = 0.036). Similar differences were found in the median times to reinfection after spontaneous clearance without treatment (365 days vs. 184 days; P = 0.01). Alternatively, among the 450 asymptomatic children with persistently positive smears, the median time for conversion to smear-negative (spontaneous clearance) was shorter for children with sickle trait than for children without (190 vs. 365 days; P = 0.02). These protective effects of sickle trait against asymptomatic P. falciparum infection under conditions of natural transmission were demonstrable using a cohort-based approach but not when the same data were examined using a cross-sectional approach.

A functional promoter variant in IL12B predisposes to cerebral malaria.

The role of the Th1 pathway in the pathogenesis of severe malaria is unclear. We recently reported that a polymorphism with increasing IFNG transcription is associated with protection against cerebral malaria (CM). Interleukin-12 is required for Th1 cell differentiation, which is characterized by the production of interferon-gamma. We investigated 21 markers in IL12-related genes, including IL12A and IL12B encoding the two IL-12 (IL12p70) subunits, IL12p35 and IL12p40. We performed a family-based association study using a total sample set of 240 nuclear families. The IL12Bpro polymorphism was associated with susceptibility to CM. The CTCTAA allele and the GC/CTCTAA genotype are over-transmitted to children with CM (P = 0.0002 and 0.00002, respectively). We estimated the odds ratio to be 2.11 for risk of CM in heterozygous children [(95% confidence interval, 1.49-2.99); P < 0.0001]. Although the CTCTAA allele had a dominant effect on CM susceptibility, this effect is much stronger in heterozygous children, consistent with the functional effects of this allele in a heterozygous form. Heterozygosity for this polymorphism has been associated with reduced expression of the gene encoding IL12p40 and a low level of IL12p70 production. These results, together with the findings from immunological studies of low interferon-gamma and IL-12 levels in CM, support a protective role for the Th1 pathway in CM.

Genetic evidence for the aggravation of Plasmodium falciparum malaria by interleukin 4.

BACKGROUND: Severe malaria (SM) due to Plasmodium falciparum causes millions of child deaths in sub-Saharan Africa. It comprises a variety of clinical disorders, including cerebral malaria (CM) and severe anemia (SA). In previous work, we have shown that interferon gamma and interleukin 12 protect against CM. Here, we investigated whether interleukin 4 (IL-4) aggravates the risk of severe disease. METHODS: We prospectively recruited children with CM (n = 240), SA (n = 101), and uncomplicated malaria (UM) (n = 42) in Bamako, Mali, and measured IL-4 production in plasma by enzyme-linked immunosorbent assay. We then assessed the influence of 11 polymorphisms on predisposition to SM by the family-based association test (FBAT). RESULTS: IL-4 concentrations were higher in children with CM than in children with UM during malaria (P = .003). FBAT analyses showed that the most significant association was between the IL4 variable-number tandem repeat (VNTR) 1/2 genotype and SM (P < .001); an association was also observed for IL4 -33 C/T, rs2243267 G/C, rs2243268 C/A, and rs2243282 C/A (P < .05). Interestingly, we found that the plasma concentration of IL-4 was higher in subjects with the IL4 VNTR 1/2 or 1/1 genotype than with the IL4 VNTR 2/2 genotype (P = .003). CONCLUSIONS: These results support the view that IL-4 may be a risk factor for SM. IL-4 may aggravate the disease by interfering with type 1 T helper cell differentiation or by promoting local inflammation at sites of parasite sequestration.

Proactive community case management and child survival: protocol for a cluster randomised controlled trial.

INTRODUCTION: Community health workers (CHWs)-shown to improve access to care and reduce maternal, newborn, and child morbidity and mortality-are re-emerging as a key strategy to achieve health-related Sustainable Development Goals (SDGs). However, recent evaluations of national programmes for CHW-led integrated community case management (iCCM) of common childhood illnesses have not found benefits on access to care and child mortality. Developing innovative ways to maximise the potential benefits of iCCM is critical to achieving the SDGs. METHODS AND ANALYSIS: An unblinded, cluster randomised controlled trial in rural Mali aims to test the efficacy of the addition of door-to-door proactive case detection by CHWs compared with a conventional approach to iCCM service delivery in reducing under-five mortality. In the intervention arm, 69 village clusters will have CHWs who conduct daily proactive case-finding home visits and deliver doorstep counsel, care, referral and follow-up. In the control arm, 68 village clusters will have CHWs who provide the same services exclusively out of a fixed community health site. A baseline population census will be conducted of all people living in the study area. All women of reproductive age will be enrolled in the study and surveyed at baseline, 12, 24 and 36 months. The survey includes a life table tracking all live births and deaths occurring prior to enrolment through the 36 months of follow-up in order to measure the primary endpoint: under-five mortality, measured as deaths among children under 5 years of age per 1000 person-years at risk of mortality. ETHICS AND DISSEMINATION: The trial has received ethical approval from the Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry, University of Bamako. The results will be disseminated through peer-reviewed publications, national and international conferences and workshops, and media outlets. TRIAL REGISTRATION NUMBER: NCT02694055; Pre-results.

Life-threatening malaria in African children: a prospective study in a mesoendemic urban setting.

BACKGROUND: The population exposed to malaria within African cities has steadily increased. However, comprehensive data on life-threatening malaria features and risk factors in children from urban areas with seasonal malaria transmission, such as in Bamako (Mali), are lacking. METHODS: Children admitted to the Gabriel Touré Hospital in Bamako with severe malarial anemia (SMA) and/or cerebral malaria (CM) were prospectively included in the study. Indicators of either SMA or CM were analyzed using logistic regression; and death hazard ratios (HRs) were estimated through survival analysis. RESULTS: The study included 455 children: 66% presented with CM, 34% with SMA, 3% with hypoglycemia (HG); 5% with dehydration; 17% with respiratory distress (RD); 25% with splenomegaly; and 92% with hepatomegaly. The children with CM were older than those with SMA. CM was more often associated with dehydration, HG, and RD, whereas SMA was more often associated with splenomegaly. The overall case fatality rate was 16%, and 94% of the children who died had CM. HG [HR: 2.37; 95% confidence interval (CI): 1.04-5.39; P = 0.040], RD (HR: 4.23; 95% CI: 2.46-7.30; P < 10(-6)) and a deep coma with a Blantyre score of less than 3 (HR: 6.78, 95% CI: 2.43-18.91; P < 10(-3)), were all independent predictors of death. CONCLUSIONS: These findings delineate the patterns of severe malaria in children in a West African mesoendemic urban setting. They validate practicable prognostic indicators of life-threatening malaria for use in the limited facilities available in African health centers and provide a frame of reference for further research addressing life-threatening malaria in this setting.

Proactive community case management and child survival in periurban Mali.

The majority of the world's population lives in urban areas, and regions with the highest under-five mortality rates are urbanising rapidly. This 7-year interrupted time series study measured early access to care and under-five mortality over the course of a proactive community case management (ProCCM) intervention in periurban Mali. Using a cluster-based, population-weighted sampling methodology, we conducted independent cross-sectional household surveys at baseline and at 12, 24, 36, 48, 60, 72 and 84 months later in the intervention area. The ProCCM intervention had five key components: (1) active case detection by community health workers (CHWs), (2) CHW doorstep care, (3) monthly dedicated supervision for CHWs, (4) removal of user fees and (5) primary care infrastructure improvements and staff capacity building. Under-five mortality rate was calculated using a Cox proportional hazard survival regression. We measured the percentage of children initiating effective antimalarial treatment within 24 hours of symptom onset and the percentage of children reported to be febrile within the previous 2 weeks. During the intervention, the rate of early effective antimalarial treatment of children 0-59 months more than doubled, from 14.7% in 2008 to 35.3% in 2015 (OR 3.198, P<0.0001). The prevalence of febrile illness among children under 5 years declined after 7 years of the intervention from 39.7% at baseline to 22.6% in 2015 (OR 0.448, P<0.0001). Communities where ProCCM was implemented have achieved an under-five mortality rate at or below 28/1000 for the past 6 years. In 2015, under-five mortality was 7/1000 (HR 0.039, P<0.0001). Further research is needed to elucidate the mechanisms of action and generalizability of ProCCM.

A Functional IL22 Polymorphism (rs2227473) Is Associated with Predisposition to Childhood Cerebral Malaria.

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection. This encephalopathy is characterized by coma and is thought to result from mechanical microvessel obstruction and an excessive activation of immune cells leading to pathological inflammation and blood-brain barrier alterations. IL-22 contributes to both chronic inflammatory and infectious diseases, and may have protective or pathogenic effects, depending on the tissue and disease state. We evaluated whether polymorphisms (n = 46) of IL22 and IL22RA2 were associated with CM in children from Nigeria and Mali. Two SNPs of IL22, rs1012356 (P = 0.016, OR = 2.12) and rs2227476 (P = 0.007, OR = 2.08) were independently associated with CM in a sample of 115 Nigerian children with CM and 160 controls. The association with rs2227476 (P = 0.01) was replicated in 240 nuclear families with one affected child from Mali. SNP rs2227473, in linkage disequilibrium with rs2227476, was also associated with CM in the combined cohort for these two populations, (P = 0.004, OR = 1.55). SNP rs2227473 is located within a putative binding site for the aryl hydrocarbon receptor, a master regulator of IL-22 production. Individuals carrying the aggravating T allele of rs2227473 produced significantly more IL-22 than those without this allele. Overall, these findings suggest that IL-22 is involved in the pathogenesis of CM.

Characterisation of the opposing effects of G6PD deficiency on cerebral malaria and severe malarial anaemia.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is believed to confer protection against Plasmodium falciparum malaria, but the precise nature of the protective effecthas proved difficult to define as G6PD deficiency has multiple allelic variants with different effects in males and females, and it has heterogeneous effects on the clinical outcome of P. falciparum infection. Here we report an analysis of multiple allelic forms of G6PD deficiency in a large multi-centre case-control study of severe malaria, using the WHO classification of G6PD mutations to estimate each individual's level of enzyme activity from their genotype. Aggregated across all genotypes, we find that increasing levels of G6PD deficiency are associated with decreasing risk of cerebral malaria, but with increased risk of severe malarial anaemia. Models of balancing selection based on these findings indicate that an evolutionary trade-off between different clinical outcomes of P. falciparum infection could have been a major cause of the high levels of G6PD polymorphism seen in human populations.

Space-time clustering of childhood malaria at the household level: a dynamic cohort in a Mali village.

BACKGROUND: Spatial and temporal heterogeneities in the risk of malaria have led the WHO to recommend fine-scale stratification of the epidemiological situation, making it possible to set up actions and clinical or basic researches targeting high-risk zones. Before initiating such studies it is necessary to define local patterns of malaria transmission and infection (in time and in space) in order to facilitate selection of the appropriate study population and the intervention allocation. The aim of this study was to identify, spatially and temporally, high-risk zones of malaria, at the household level (resolution of 1 to 3 m). METHODS: This study took place in a Malian village with hyperendemic seasonal transmission as part of Mali-Tulane Tropical Medicine Research Center (NIAID/NIH). The study design was a dynamic cohort (22 surveys, from June 1996 to June 2001) on about 1300 children (<12 years) distributed between 173 households localized by GPS. We used the computed parasitological data to analyzed levels of Plasmodium falciparum, P. malariae and P. ovale infection and P. falciparum gametocyte carriage by means of time series and Kulldorff's scan statistic for space-time cluster detection. RESULTS: The time series analysis determined that malaria parasitemia (primarily P. falciparum) was persistently present throughout the population with the expected seasonal variability pattern and a downward temporal trend. We identified six high-risk clusters of P. falciparum infection, some of which persisted despite an overall tendency towards a decrease in risk. The first high-risk cluster of P. falciparum infection (rate ratio = 14.161) was detected from September 1996 to October 1996, in the north of the village. CONCLUSION: This study showed that, although infection proportions tended to decrease, high-risk zones persisted in the village particularly near temporal backwaters. Analysis of this heterogeneity at the household scale by GIS methods lead to target preventive actions more accurately on the high-risk zones identified. This mapping of malaria risk makes it possible to orient control programs, treating the high-risk zones identified as a matter of priority, and to improve the planning of intervention trials or research studies on malaria.

Oblique decision trees for spatial pattern detection: optimal algorithm and application to malaria risk.

BACKGROUND: In order to detect potential disease clusters where a putative source cannot be specified, classical procedures scan the geographical area with circular windows through a specified grid imposed to the map. However, the choice of the windows' shapes, sizes and centers is critical and different choices may not provide exactly the same results. The aim of our work was to use an Oblique Decision Tree model (ODT) which provides potential clusters without pre-specifying shapes, sizes or centers. For this purpose, we have developed an ODT-algorithm to find an oblique partition of the space defined by the geographic coordinates. METHODS: ODT is based on the classification and regression tree (CART). As CART finds out rectangular partitions of the covariate space, ODT provides oblique partitions maximizing the interclass variance of the independent variable. Since it is a NP-Hard problem in RN, classical ODT-algorithms use evolutionary procedures or heuristics. We have developed an optimal ODT-algorithm in R2, based on the directions defined by each couple of point locations. This partition provided potential clusters which can be tested with Monte-Carlo inference. We applied the ODT-model to a dataset in order to identify potential high risk clusters of malaria in a village in Western Africa during the dry season. The ODT results were compared with those of the Kulldorff' s SaTScan. RESULTS: The ODT procedure provided four classes of risk of infection. In the first high risk class 60%, 95% confidence interval (CI95%) [52.22-67.55], of the children was infected. Monte-Carlo inference showed that the spatial pattern issued from the ODT-model was significant (p < 0.0001). Satscan results yielded one significant cluster where the risk of disease was high with an infectious rate of 54.21%, CI95% [47.51-60.75]. Obviously, his center was located within the first high risk ODT class. Both procedures provided similar results identifying a high risk cluster in the western part of the village where a mosquito breeding point was located. CONCLUSION: ODT-models improve the classical scanning procedures by detecting potential disease clusters independently of any specification of the shapes, sizes or centers of the clusters.

Risk factors for severe malaria in Bamako, Mali: a matched case-control study.

The aim of this case-control study was to identify epidemiological risk factors for severe malaria among children living in Bamako, a malaria-endemic area. For this, 260 healthy community controls were matched to 130 patients with severe malaria. Conditional multiple logistic regression analysis indicated that all examined independent factors associated with severe malaria are directly related to characteristics of the child's mother, with the exception of the child's own yellow fever vaccination history (odds ratio (OR): 1.93, 95% confidence intervals (CI(95%)) [1.10-3.37]). The following characteristics were all associated with a decreased risk of severe malaria in the child: maternal education (OR: 0.52, CI(95%) [0.31-0.86]), the mother's adequate knowledge about malaria (OR: 0.46, 95% CI(95%) [0.25-0.86]), her use of mosquito bed nets (OR: 0.53, CI(95%) [0.30-0.92]) and breast-feeding for at least 2 years (OR: 0.57, CI(95%) [0.33-0.94]). Conversely, chronic maternal disease (OR: ?3.16, CI(95%) [1.31-7.61]) was associated with an increased risk of severe malaria. These findings strongly support the hypothesis that maternal factors are central to the development of severe malaria in children. Programmes aiming to improve both maternal health and maternal education may reduce the incidence of severe malaria in children and should therefore be advocated in Bamako and in areas with similar epidemiological patterns for malaria.

Candidate polymorphisms and severe malaria in a Malian population.

Malaria is a major health burden in sub-Saharan African countries, including Mali. The disease is complex, with multiple genetic determinants influencing the observed variation in response to infection, progression, and severity. We assess the influence of sixty-four candidate loci, including the sickle cell polymorphism (HbS), on severe malaria in a case-control study consisting of over 900 individuals from Bamako, Mali. We confirm the known protective effects of the blood group O and the HbS AS genotype on life-threatening malaria. In addition, our analysis revealed a marginal susceptibility effect for the CD40 ligand (CD40L)+220C allele. The lack of statistical evidence for other candidates may demonstrate the need for large-scale genome-wide association studies in malaria to discover new polymorphisms. It also demonstrates the need for establishing the region-specific repertoire of functional variation in important genes, including the glucose-6-phosphatase deficiency gene, before embarking on focused genotyping.

Sahel, savana, riverine and urban malaria in West Africa: Similar control policies with different outcomes.

The study sites for the West African ICEMR are in three countries (The Gambia, Senegal, Mali) and are located within 750 km of each other. In addition, the National Malaria Control Programmes of these countries have virtually identical policies: (1) Artemisinin Combination Therapies (ACTs) for the treatment of symptomatic Plasmodium falciparum infection, (2) Long-Lasting Insecticide-treated bed Nets (LLINs) to reduce the Entomololgic Inoculation Rate (EIR), and (3) sulfadoxine-pyrimethamine for the Intermittent Preventive Treatment of malaria during pregnancy (IPTp). However, the prevalence of P. falciparum malaria and the status of malaria control vary markedly across the four sites with differences in the duration of the transmission season (from 4-5 to 10-11 months), the intensity of transmission (with EIRs from unmeasurably low to 4-5 per person per month), multiplicity of infection (from a mean of 1.0 to means of 2-5) and the status of malaria control (from areas which have virtually no control to areas that are at the threshold of malaria elimination). The most important priority is the need to obtain comparable data on the population-based prevalence, incidence and transmission of malaria before new candidate interventions or combinations of interventions are introduced for malaria control.

Improving malaria control in West Africa: interruption of transmission as a paradigm shift.

With the paradigm shift from the reduction of morbidity and mortality to the interruption of transmission, the focus of malaria control broadens from symptomatic infections in children ≤5 years of age to include asymptomatic infections in older children and adults. In addition, as control efforts intensify and the number of interventions increases, there will be decreases in prevalence, incidence and transmission with additional decreases in morbidity and mortality. Expected secondary consequences of these changes include upward shifts in the peak ages for infection (parasitemia) and disease, increases in the ages for acquisition of antiparasite humoral and cellular immune responses and increases in false-negative blood smears and rapid diagnostic tests. Strategies to monitor these changes must include: (1) studies of the entire population (that are not restricted to children ≤5 or ≤10 years of age), (2) study sites in both cities and rural areas (because of increasing urbanization across sub-Saharan Africa) and (3) innovative strategies for surveillance as the prevalence of infection decreases and the frequency of false-negative smears and rapid diagnostic tests increases.