Non-O ABO blood group genotypes differ in their associations with Plasmodium falciparum rosetting and severe malaria.

Blood group O is associated with protection against severe malaria and reduced size and stability of P. falciparum-host red blood cell (RBC) rosettes compared to non-O blood groups. Whether the non-O blood groups encoded by the specific ABO genotypes AO, BO, AA, BB and AB differ in their associations with severe malaria and rosetting is unknown. The A and B antigens are host RBC receptors for rosetting, hence we hypothesized that the higher levels of A and/or B antigen on RBCs from AA, BB and AB genotypes compared to AO/BO genotypes could lead to larger rosettes, increased microvascular obstruction and higher risk of malaria pathology. We used a case-control study of Kenyan children and in vitro adhesion assays to test the hypothesis that "double dose" non-O genotypes (AA, BB, AB) are associated with increased risk of severe malaria and larger rosettes than "single dose" heterozygotes (AO, BO). In the case-control study, compared to OO, the double dose genotypes consistently had higher odds ratios (OR) for severe malaria than single dose genotypes, with AB (OR 1.93) and AO (OR 1.27) showing most marked difference (p = 0.02, Wald test). In vitro experiments with blood group A-preferring P. falciparum parasites showed that significantly larger rosettes were formed with AA and AB host RBCs compared to OO, whereas AO and BO genotypes rosettes were indistinguishable from OO. Overall, the data show that ABO genotype influences P. falciparum rosetting and support the hypothesis that double dose non-O genotypes confer a greater risk of severe malaria than AO/BO heterozygosity.

Genome-wide association study of leprosy in Malawi and Mali.

Leprosy is a chronic infection of the skin and peripheral nerves caused by Mycobacterium leprae. Despite recent improvements in disease control, leprosy remains an important cause of infectious disability globally. Large-scale genetic association studies in Chinese, Vietnamese and Indian populations have identified over 30 susceptibility loci for leprosy. There is a significant burden of leprosy in Africa, however it is uncertain whether the findings of published genetic association studies are generalizable to African populations. To address this, we conducted a genome-wide association study (GWAS) of leprosy in Malawian (327 cases, 436 controls) and Malian (247 cases, 368 controls) individuals. In that analysis, we replicated four risk loci previously reported in China, Vietnam and India; MHC Class I and II, LACC1 and SLC29A3. We further identified a novel leprosy susceptibility locus at 10q24 (rs2015583; combined p = 8.81 × 10-9; OR = 0.51 [95% CI 0.40 - 0.64]). Using publicly-available data we characterise regulatory activity at this locus, identifying ACTR1A as a candidate mediator of leprosy risk. This locus shows evidence of recent positive selection and demonstrates pleiotropy with established risk loci for inflammatory bowel disease and childhood-onset asthma. A shared genetic architecture for leprosy and inflammatory bowel disease has been previously described. We expand on this, strengthening the hypothesis that selection pressure driven by leprosy has shaped the evolution of autoimmune and atopic disease in modern populations. More broadly, our data highlights the importance of defining the genetic architecture of disease across genetically diverse populations, and that disease insights derived from GWAS in one population may not translate to all affected populations.

BIRC6 modifies risk of invasive bacterial infection in Kenyan children.

Invasive bacterial disease is a major cause of morbidity and mortality in African children. Despite being caused by diverse pathogens, children with sepsis are clinically indistinguishable from one another. In spite of this, most genetic susceptibility loci for invasive infection that have been discovered to date are pathogen specific and are not therefore suggestive of a shared genetic architecture of bacterial sepsis. Here, we utilise probabilistic diagnostic models to identify children with a high probability of invasive bacterial disease among critically unwell Kenyan children with Plasmodium falciparum parasitaemia. We construct a joint dataset including 1445 bacteraemia cases and 1143 severe malaria cases, and population controls, among critically unwell Kenyan children that have previously been genotyped for human genetic variation. Using these data, we perform a cross-trait genome-wide association study of invasive bacterial infection, weighting cases according to their probability of bacterial disease. In doing so, we identify and validate a novel risk locus for invasive infection secondary to multiple bacterial pathogens, that has no apparent effect on malaria risk. The locus identified modifies splicing of BIRC6 in stimulated monocytes, implicating regulation of apoptosis and autophagy in the pathogenesis of sepsis in Kenyan children.

Bacterial infections are a major cause of severe illness and death in African children. Understanding which children are at risk of life-threatening infection and why, is key to designing new tools to help protect them. Some risk is likely inherited, but scientists do not know which genes are responsible. Genome-wide association studies (GWAS) may be one way to identify bacterial infection risk genes. GWAS look for genetic differences associated with a particular disease. But previous GWAS studies have failed to find genes linked with bacterial infections in African children because they were too small. Malaria is another frequent cause of life-threatening illness in African children. It can be hard for clinicians to determine if a child's illness is caused by malaria, a bacterial infection, or both. Many children in Africa have malaria parasites in their blood, but they do not always cause disease. Most children with suspected severe malaria are treated with antibiotics in case of bacterial infection. Clinicians may then conduct further testing to determine the illness's actual cause. Scientists may be able to use this data on children with suspected malaria to study bacterial infections. Gilchrist et al. show that children with an unusual alteration in the BIRC6 gene are at increased risk of bacterial infections. In the experiments, Gilchrist et al. used computer modeling to identify a subset of children with likely bacterial infections among 2,200 children admitted to a hospital in Kenya with a high fever and malaria parasites. By combining information on this subset of children with data on children with confirmed bacterial infections and healthy children, Gilchrist created a sample of 5,400 children for a GWAS. The analyses found that children with a variation in the BIRC6 gene on chromosome 2 had a higher risk of bacterial infections. This genetic change is linked with the production of a modified form of BIRC6 in infection-fighting immune cells called monocytes. More studies will help scientists understand how this change might contribute to severe bacterial infections. Learning more may help scientists develop new treatment strategies and identify children most at risk.

Malaria protection due to sickle haemoglobin depends on parasite genotype.

Host genetic factors can confer resistance against malaria1, raising the question of whether this has led to evolutionary adaptation of parasite populations. Here we searched for association between candidate host and parasite genetic variants in 3,346 Gambian and Kenyan children with severe malaria caused by Plasmodium falciparum. We identified a strong association between sickle haemoglobin (HbS) in the host and three regions of the parasite genome, which is not explained by population structure or other covariates, and which is replicated in additional samples. The HbS-associated alleles include nonsynonymous variants in the gene for the acyl-CoA synthetase family member2-4 PfACS8 on chromosome 2, in a second region of chromosome 2, and in a region containing structural variation on chromosome 11. The alleles are in strong linkage disequilibrium and have frequencies that covary with the frequency of HbS across populations, in particular being much more common in Africa than other parts of the world. The estimated protective effect of HbS against severe malaria, as determined by comparison of cases with population controls, varies greatly according to the parasite genotype at these three loci. These findings open up a new avenue of enquiry into the biological and epidemiological significance of the HbS-associated polymorphisms in the parasite genome and the evolutionary forces that have led to their high frequency and strong linkage disequilibrium in African P. falciparum populations.

Temporal evolution of sulfadoxine-pyrimethamine resistance genotypes and genetic diversity in response to a decade of increased interventions against Plasmodium falciparum in northern Ghana.

BACKGROUND: Anti-malarial drug resistance remains a key concern for the global fight against malaria. In Ghana sulfadoxine-pyrimethamine (SP) is used for intermittent preventive treatment of malaria in pregnancy and combined with amodiaquine for Seasonal Malaria Chemoprevention (SMC) during the high malaria season. Thus, surveillance of molecular markers of SP resistance is important to guide decision-making for these interventions in Ghana. METHODS: A total of 4469 samples from uncomplicated malaria patients collected from 2009 to 2018 was submitted to the Wellcome Trust Sanger Institute, UK for DNA sequencing using MiSeq. Genotypes were successfully translated into haplotypes in 2694 and 846 mono infections respectively for pfdhfr and pfdhps genes and the combined pfhdfr/pfdhps genes across all years. RESULTS: At the pfdhfr locus, a consistently high (> 60%) prevalence of parasites carrying triple mutants (IRNI) were detected from 2009 to 2018. Two double mutant haplotypes (NRNI and ICNI) were found, with haplotype NRNI having a much higher prevalence (average 13.8%) than ICNI (average 3.2%) across all years. Six pfdhps haplotypes were detected. Of these, prevalence of five fluctuated in a downward trend over time from 2009 to 2018, except a pfdhps double mutant (AGKAA), which increased consistently from 2.5% in 2009 to 78.2% in 2018. Across both genes, pfdhfr/pfdhps combined triple (NRNI + AAKAA) mutants were only detected in 2009, 2014, 2015 and 2018, prevalence of which fluctuated between 3.5 and 5.5%. The combined quadruple (IRNI + AAKAA) genotype increased in prevalence from 19.3% in 2009 to 87.5% in 2011 before fluctuating downwards to 19.6% in 2018 with an average prevalence of 37.4% within the nine years. Prevalence of parasites carrying the quintuple (IRNI + AGKAA or SGEAA) mutant haplotypes, which are highly refractory to SP increased over time from 14.0% in 2009 to 89.0% in 2016 before decreasing to 78.9 and 76.6% in 2017 and 2018 respectively. Though quintuple mutants are rising in prevalence in both malaria seasons, together these combined genotypes vary significantly within season but not between seasons. CONCLUSIONS: Despite high prevalence of pfdhfr triple mutants and combined pfdhfr/pfdhps quadruple and quintuple mutants in this setting SP may still be efficacious. These findings are significant as they highlight the need to continuously monitor SP resistance, particularly using deep targeted sequencing to ascertain changing resistance patterns.

Malaria is a cause of iron deficiency in African children.

Malaria and iron deficiency (ID) are common and interrelated public health problems in African children. Observational data suggest that interrupting malaria transmission reduces the prevalence of ID1. To test the hypothesis that malaria might cause ID, we used sickle cell trait (HbAS, rs334 ), a genetic variant that confers specific protection against malaria2, as an instrumental variable in Mendelian randomization analyses. HbAS was associated with a 30% reduction in ID among children living in malaria-endemic countries in Africa (n = 7,453), but not among individuals living in malaria-free areas (n = 3,818). Genetically predicted malaria risk was associated with an odds ratio of 2.65 for ID per unit increase in the log incidence rate of malaria. This suggests that an intervention that halves the risk of malaria episodes would reduce the prevalence of ID in African children by 49%.

Novel genotyping approaches to easily detect genomic admixture between the major Afrotropical malaria vector species, Anopheles coluzzii and An. gambiae.

The two most efficient and most recently radiated Afrotropical vectors of human malaria - Anopheles coluzzii and An. gambiae - are identified by single-locus diagnostic PCR assays based on species-specific markers in a 4 Mb region on chromosome-X centromere. Inherently, these diagnostic assays cannot detect interspecific autosomal admixture shown to be extensive at the westernmost and easternmost extremes of the species range. The main aim of this study was to develop novel, easy-to-implement tools for genotyping An. coluzzii and An. gambiae-specific ancestral informative markers (AIMs) identified from the Anopheles gambiae 1000 genomes (Ag1000G) project. First, we took advantage of this large set of data in order to develop a multilocus approach to genotype 26 AIMs on all chromosome arms valid across the species range. Second, we tested the multilocus assay on samples from Guinea Bissau, The Gambia and Senegal, three countries spanning the westernmost hybridization zone, where conventional species diagnostic is problematic due to the putative presence of a novel "hybrid form". The multilocus assay was able to capture patterns of admixture reflecting those revealed by the whole set of AIMs and provided new original data on interspecific admixture in the region. Third, we developed an easy-to-use, cost-effective PCR approach for genotyping two AIMs on chromosome-3 among those included in the multilocus approach, opening the possibility for advanced identification of species and of admixed specimens during routine large scale entomological surveys, particularly, but not exclusively, at the extremes of the range, where WGS data highlighted unexpected autosomal admixture.

High-throughput genotyping assays for identification of glycophorin B deletion variants in population studies.

Glycophorins are the most abundant sialoglycoproteins on the surface of human erythrocyte membranes. Genetic variation in glycophorin region of human chromosome 4 (containing GYPA, GYPB, and GYPE genes) is of interest because the gene products serve as receptors for pathogens of major public health interest, including Plasmodiumsp., Babesiasp., Influenza virus, Vibrio cholerae El Tor Hemolysin, and Escherichia coli. A large structural rearrangement and hybrid glycophorin variant, known as Dantu, which was identified in East African populations, has been linked with a 40% reduction in risk for severe malaria. Apart from Dantu, other large structural variants exist, with the most common being deletion of the whole GYPB gene and its surrounding region, resulting in multiple different deletion forms. In West Africa particularly, these deletions are estimated to account for between 5 and 15% of the variation in different populations, mostly attributed to the forms known as DEL1 and DEL2. Due to the lack of specific variant assays, little is known of the distribution of these variants. Here, we report a modification of a previous GYPB DEL1 assay and the development of a novel GYPB DEL2 assay as high-throughput PCR-RFLP assays, as well as the identification of the crossover/breakpoint for GYPB DEL2. Using 393 samples from three study sites in Ghana as well as samples from HapMap and 1000 G projects for validation, we show that our assays are sensitive and reliable for genotyping GYPB DEL1 and DEL2. To the best of our knowledge, this is the first report of such high-throughput genotyping assays by PCR-RFLP for identifying specific GYPB deletion types in populations. These assays will enable better identification of GYPB deletions for large genetic association studies and functional experiments to understand the role of this gene cluster region in susceptibility to malaria and other diseases.

Interferon-gamma polymorphisms and risk of iron deficiency and anaemia in Gambian children.

Background: Anaemia is a major public health concern especially in African children living in malaria-endemic regions. Interferon-gamma (IFN-γ) is elevated during malaria infection and is thought to influence erythropoiesis and iron status. Genetic variants in the IFN-γ gene (IFNG) are associated with increased IFN-γ production. We investigated putative functional single nucleotide polymorphisms (SNPs) and haplotypes of IFNG in relation to nutritional iron status and anaemia in Gambian children over a malaria season. Methods: We used previously available data from Gambian family trios to determine informative SNPs and then used the Agena Bioscience MassArray platform to type five SNPs from the IFNG gene in a cohort of 780 Gambian children aged 2-6 years. We also measured haemoglobin and biomarkers of iron status and inflammation at the start and end of a malaria season. Results: We identified five IFNG haplotype-tagging SNPs ( IFNG-1616 [rs2069705], IFNG+874 [rs2430561], IFNG+2200 [rs1861493], IFNG+3234 [rs2069718] and IFNG+5612 [rs2069728]). The IFNG+2200C [rs1861493] allele was associated with reduced haemoglobin concentrations (adjusted ß -0.44 [95% CI -0.75, -0.12]; Bonferroni adjusted P = 0.03) and a trend towards iron deficiency compared to wild-type at the end of the malaria season in multivariable models adjusted for potential confounders. A haplotype uniquely identified by IFNG+2200C was similarly associated with reduced haemoglobin levels and trends towards iron deficiency, anaemia and iron deficiency anaemia at the end of the malaria season in models adjusted for age, sex, village, inflammation and malaria parasitaemia. Conclusion: We found limited statistical evidence linking IFNG polymorphisms with a risk of developing iron deficiency and anaemia in Gambian children. More definitive studies are needed to investigate the effects of genetically influenced IFN-γ levels on the risk of iron deficiency and anaemia in children living in malaria-endemic areas.

Haplotype heterogeneity and low linkage disequilibrium reduce reliable prediction of genotypes for the ­α 3.7I form of α-thalassaemia using genome-wide microarray data.

Background: The -α 3.7I-thalassaemia deletion is very common throughout Africa because it protects against malaria. When undertaking studies to investigate human genetic adaptations to malaria or other diseases, it is important to account for any confounding effects of α-thalassaemia to rule out spurious associations. Methods: In this study we have used direct α-thalassaemia genotyping to understand why GWAS data from a large malaria association study in Kilifi Kenya did not identify the α-thalassaemia signal. We then explored the potential use of a number of new approaches to using GWAS data for imputing α-thalassaemia as an alternative to direct genotyping by PCR. Results: We found very low linkage-disequilibrium of the directly typed data with the GWAS SNP markers around α-thalassaemia and across the haemoglobin-alpha ( HBA) gene region, which along with a complex haplotype structure, could explain the lack of an association signal from the GWAS SNP data. Some indirect typing methods gave results that were in broad agreement with those derived from direct genotyping and could identify an association signal, but none were sufficiently accurate to allow correct interpretation compared with direct typing, leading to confusing or erroneous results. Conclusions: We conclude that going forwards, direct typing methods such as PCR will still be required to account for α-thalassaemia in GWAS studies.

A high throughput multi-locus insecticide resistance marker panel for tracking resistance emergence and spread in Anopheles gambiae.

The spread of resistance to insecticides in disease-carrying mosquitoes poses a threat to the effectiveness of control programmes, which rely largely on insecticide-based interventions. Monitoring mosquito populations is essential, but obtaining phenotypic measurements of resistance is laborious and error-prone. High-throughput genotyping offers the prospect of quick and repeatable estimates of resistance, while also allowing resistance markers to be tracked and studied. To demonstrate the potential of highly-mulitplexed genotypic screening for measuring resistance-association of mutations and tracking their spread, we developed a panel of 28 known or putative resistance markers in the major malaria vector Anopheles gambiae, which we used to screen mosquitoes from a wide swathe of Sub-Saharan Africa (Burkina Faso, Ghana, Democratic Republic of Congo (DRC) and Kenya). We found resistance association in four markers, including a novel mutation in the detoxification gene Gste2 (Gste2-119V). We also identified a duplication in Gste2 combining a resistance-associated mutation with its wild-type counterpart, potentially alleviating the costs of resistance. Finally, we describe the distribution of the multiple origins of kdr resistance, finding unprecedented diversity in the DRC. This panel represents the first step towards a quantitative genotypic model of insecticide resistance that can be used to predict resistance status in An. gambiae.

Human candidate gene polymorphisms and risk of severe malaria in children in Kilifi, Kenya: a case-control association study.

BACKGROUND: Human genetic factors are important determinants of malaria risk. We investigated associations between multiple candidate polymorphisms-many related to the structure or function of red blood cells-and risk for severe Plasmodium falciparum malaria and its specific phenotypes, including cerebral malaria, severe malaria anaemia, and respiratory distress. METHODS: We did a case-control study in Kilifi County, Kenya. We recruited as cases children presenting with severe malaria to the high-dependency ward of Kilifi County Hospital. We included as controls infants born in the local community between Aug 1, 2006, and Sept 30, 2010, who were part of a genetics study. We tested for associations between a range of candidate malaria-protective genes and risk for severe malaria and its specific phenotypes. We used a permutation approach to account for multiple comparisons between polymorphisms and severe malaria. We judged p values less than 0·005 significant for the primary analysis of the association between candidate genes and severe malaria. FINDINGS: Between June 11, 1995, and June 12, 2008, 2244 children with severe malaria were recruited to the study, and 3949 infants were included as controls. Overall, 263 (12%) of 2244 children with severe malaria died in hospital, including 196 (16%) of 1233 with cerebral malaria. We investigated 121 polymorphisms in 70 candidate severe malaria-associated genes. We found significant associations between risk for severe malaria overall and polymorphisms in 15 genes or locations, of which most were related to red blood cells: ABO, ATP2B4, ARL14, CD40LG, FREM3, INPP4B, G6PD, HBA (both HBA1 and HBA2), HBB, IL10, LPHN2 (also known as ADGRL2), LOC727982, RPS6KL1, CAND1, and GNAS. Combined, these genetic associations accounted for 5·2% of the variance in risk for developing severe malaria among individuals in the general population. We confirmed established associations between severe malaria and sickle-cell trait (odds ratio [OR] 0·15, 95% CI 0·11-0·20; p=2·61 × 10-58), blood group O (0·74, 0·66-0·82; p=6·26 × 10-8), and -α3·7-thalassaemia (0·83, 0·76-0·90; p=2·06 × 10-6). We also found strong associations between overall risk of severe malaria and polymorphisms in both ATP2B4 (OR 0·76, 95% CI 0·63-0·92; p=0·001) and FREM3 (0·64, 0·53-0·79; p=3·18 × 10-14). The association with FREM3 could be accounted for by linkage disequilibrium with a complex structural mutation within the glycophorin gene region (comprising GYPA, GYPB, and GYPE) that encodes for the rare Dantu blood group antigen. Heterozygosity for Dantu was associated with risk for severe malaria (OR 0·57, 95% CI 0·49-0·68; p=3·22 × 10-11), as was homozygosity (0·26, 0·11-0·62; p=0·002). INTERPRETATION: Both ATP2B4 and the Dantu blood group antigen are associated with the structure and function of red blood cells. ATP2B4 codes for plasma membrane calcium-transporting ATPase 4 (the major calcium pump on red blood cells) and the glycophorins are ligands for parasites to invade red blood cells. Future work should aim at uncovering the mechanisms by which these polymorphisms can result in severe malaria protection and investigate the implications of these associations for wider health. FUNDING: Wellcome Trust, UK Medical Research Council, European Union, and Foundation for the National Institutes of Health as part of the Bill & Melinda Gates Grand Challenges in Global Health Initiative.

Two complement receptor one alleles have opposing associations with cerebral malaria and interact with α+thalassaemia.

Malaria has been a major driving force in the evolution of the human genome. In sub-Saharan African populations, two neighbouring polymorphisms in the Complement Receptor One (CR1) gene, named Sl2 and McCb, occur at high frequencies, consistent with selection by malaria. Previous studies have been inconclusive. Using a large case-control study of severe malaria in Kenyan children and statistical models adjusted for confounders, we estimate the relationship between Sl2 and McCb and malaria phenotypes, and find they have opposing associations. The Sl2 polymorphism is associated with markedly reduced odds of cerebral malaria and death, while the McCb polymorphism is associated with increased odds of cerebral malaria. We also identify an apparent interaction between Sl2 and α+thalassaemia, with the protective association of Sl2 greatest in children with normal α-globin. The complex relationship between these three mutations may explain previous conflicting findings, highlighting the importance of considering genetic interactions in disease-association studies.

Malaria Host Candidate Genes Validated by Association With Current, Recent, and Historical Measures of Transmission Intensity.

Background: Human malaria susceptibility is determined by multiple genetic factors. It is unclear, however, which genetic variants remain important over time. Methods: Genetic associations of 175 high-quality polymorphisms within several malaria candidate genes were examined in a sample of 8096 individuals from northeast Tanzania using altitude, seroconversion rates, and parasite rates as proxies of historical, recent, and current malaria transmission intensity. A principal component analysis was used to derive 2 alternative measures of overall malaria propensity of a location across different time scales. Results: Common red blood cell polymorphisms (ie, hemoglobin S, glucose-6-phosphate dehydrogenase, and α-thalassemia) were the only ones to be associated with all 3 measures of transmission intensity and the first principal component. Moderate associations were found between some immune response genes (ie, IL3 and IL13) and parasite rates, but these could not be reproduced using the alternative measures of malaria propensity. Conclusions: We have demonstrated the potential of using altitude and seroconversion rate as measures of malaria transmission capturing medium- to long-term time scales to detect genetic associations that are likely to persist over time. These measures also have the advantage of minimizing the deleterious effects of random factors affecting parasite rates on the respective association signals.

Resistance to malaria through structural variation of red blood cell invasion receptors.

The malaria parasite Plasmodium falciparum invades human red blood cells by a series of interactions between host and parasite surface proteins. By analyzing genome sequence data from human populations, including 1269 individuals from sub-Saharan Africa, we identify a diverse array of large copy-number variants affecting the host invasion receptor genes GYPA and GYPB We find that a nearby association with severe malaria is explained by a complex structural rearrangement involving the loss of GYPB and gain of two GYPB-A hybrid genes, which encode a serologically distinct blood group antigen known as Dantu. This variant reduces the risk of severe malaria by 40% and has recently increased in frequency in parts of Kenya, yet it appears to be absent from west Africa. These findings link structural variation of red blood cell invasion receptors with natural resistance to severe malaria.

THE REAL McCOIL: A method for the concurrent estimation of the complexity of infection and SNP allele frequency for malaria parasites.

As many malaria-endemic countries move towards elimination of Plasmodium falciparum, the most virulent human malaria parasite, effective tools for monitoring malaria epidemiology are urgent priorities. P. falciparum population genetic approaches offer promising tools for understanding transmission and spread of the disease, but a high prevalence of multi-clone or polygenomic infections can render estimation of even the most basic parameters, such as allele frequencies, challenging. A previous method, COIL, was developed to estimate complexity of infection (COI) from single nucleotide polymorphism (SNP) data, but relies on monogenomic infections to estimate allele frequencies or requires external allele frequency data which may not available. Estimates limited to monogenomic infections may not be representative, however, and when the average COI is high, they can be difficult or impossible to obtain. Therefore, we developed THE REAL McCOIL, Turning HEterozygous SNP data into Robust Estimates of ALelle frequency, via Markov chain Monte Carlo, and Complexity Of Infection using Likelihood, to incorporate polygenomic samples and simultaneously estimate allele frequency and COI. This approach was tested via simulations then applied to SNP data from cross-sectional surveys performed in three Ugandan sites with varying malaria transmission. We show that THE REAL McCOIL consistently outperforms COIL on simulated data, particularly when most infections are polygenomic. Using field data we show that, unlike with COIL, we can distinguish epidemiologically relevant differences in COI between and within these sites. Surprisingly, for example, we estimated high average COI in a peri-urban subregion with lower transmission intensity, suggesting that many of these cases were imported from surrounding regions with higher transmission intensity. THE REAL McCOIL therefore provides a robust tool for understanding the molecular epidemiology of malaria across transmission settings.

Severe anemia in Malawian children.

BACKGROUND: Severe anemia is a major cause of sickness and death in African children, yet the causes of anemia in this population have been inadequately studied. METHODS: We conducted a case-control study of 381 preschool children with severe anemia (hemoglobin concentration, <5.0 g per deciliter) and 757 preschool children without severe anemia in urban and rural settings in Malawi. Causal factors previously associated with severe anemia were studied. The data were examined by multivariate analysis and structural equation modeling. RESULTS: Bacteremia (adjusted odds ratio, 5.3; 95% confidence interval [CI], 2.6 to 10.9), malaria (adjusted odds ratio, 2.3; 95% CI, 1.6 to 3.3), hookworm (adjusted odds ratio, 4.8; 95% CI, 2.0 to 11.8), human immunodeficiency virus infection (adjusted odds ratio, 2.0; 95% CI, 1.0 to 3.8), the G6PD-202/-376 genetic disorder (adjusted odds ratio, 2.4; 95% CI, 1.3 to 4.4), vitamin A deficiency (adjusted odds ratio, 2.8; 95% CI, 1.3 to 5.8), and vitamin B12 deficiency (adjusted odds ratio, 2.2; 95% CI, 1.4 to 3.6) were associated with severe anemia. Folate deficiency, sickle cell disease, and laboratory signs of an abnormal inflammatory response were uncommon. Iron deficiency was not prevalent in case patients (adjusted odds ratio, 0.37; 95% CI, 0.22 to 0.60) and was negatively associated with bacteremia. Malaria was associated with severe anemia in the urban site (with seasonal transmission) but not in the rural site (where malaria was holoendemic). Seventy-six percent of hookworm infections were found in children under 2 years of age. CONCLUSIONS: There are multiple causes of severe anemia in Malawian preschool children, but folate and iron deficiencies are not prominent among them. Even in the presence of malaria parasites, additional or alternative causes of severe anemia should be considered.

Genomic analysis of local variation and recent evolution in Plasmodium vivax.

The widespread distribution and relapsing nature of Plasmodium vivax infection present major challenges for the elimination of malaria. To characterize the genetic diversity of this parasite in individual infections and across the population, we performed deep genome sequencing of >200 clinical samples collected across the Asia-Pacific region and analyzed data on >300,000 SNPs and nine regions of the genome with large copy number variations. Individual infections showed complex patterns of genetic structure, with variation not only in the number of dominant clones but also in their level of relatedness and inbreeding. At the population level, we observed strong signals of recent evolutionary selection both in known drug resistance genes and at new loci, and these varied markedly between geographical locations. These findings demonstrate a dynamic landscape of local evolutionary adaptation in the parasite population and provide a foundation for genomic surveillance to guide effective strategies for control and elimination of P. vivax.

Admixture into and within sub-Saharan Africa.

Similarity between two individuals in the combination of genetic markers along their chromosomes indicates shared ancestry and can be used to identify historical connections between different population groups due to admixture. We use a genome-wide, haplotype-based, analysis to characterise the structure of genetic diversity and gene-flow in a collection of 48 sub-Saharan African groups. We show that coastal populations experienced an influx of Eurasian haplotypes over the last 7000 years, and that Eastern and Southern Niger-Congo speaking groups share ancestry with Central West Africans as a result of recent population expansions. In fact, most sub-Saharan populations share ancestry with groups from outside of their current geographic region as a result of gene-flow within the last 4000 years. Our in-depth analysis provides insight into haplotype sharing across different ethno-linguistic groups and the recent movement of alleles into new environments, both of which are relevant to studies of genetic epidemiology.

Tumour necrosis factor alpha promoter polymorphism, TNF-238 is associated with severe clinical outcome of falciparum malaria in Ibadan southwest Nigeria.

Tumour necrosis factor (TNF) - α has been shown to play an important role in the pathogenesis of falciparum malaria. Two TNF promoter polymorphisms, TNF-308 and TNF-238 have been associated with differential activity and production of TNF. In order to investigate the association between TNF-308 and TNF-238 and the clinical outcome of malaria in a Nigerian population, the two TNF polymorphisms were analysed using Sequenom iPLEX Platform. A total of 782 children; 283 children with uncomplicated malaria, 255 children with severe malaria and 244 children with asymptomatic infection (controls) were studied. The distribution of TNF-308 and TNF-238 genotypes were consistent with the Hardy-Weinberg equilibrium. Distribution of both TNF polymorphisms differed significantly across all clinical groups (TNF-308: p=0.007; TNF-238: p=0.001). Further tests for association with severe malaria using genotype models controlling for age, parasitaemia and HbAS showed a significant association of the TNF-238 polymorphism with susceptibility to severe malaria (95% CI=1.43-6.02, OR=2.94, p=0.003237) The GG genotype of TNF-238 significantly increased the risk of developing cerebral malaria from asymptomatic malaria and uncomplicated malaria (95% CI=1.99-18.17, OR=6.02, p<0.001 and 95% CI=1.78-8.23, OR=3.84, p<0.001 respectively). No significant association was found between TNF-308 and malaria outcome. These results show thegenetic association of TNF-238 in the clinical outcome of malaria in Ibadan, southwest Nigeria. These findings add support to the role of TNF in the outcome of malaria infection. Further large scale studies across multiple malaria endemic populations will be required to determine the specific roles of TNF-308 and TNF-238 in the outcome of falciparum malaria infection.