BACKGROUND: Plasmodium falciparum erythrocyte membrane protein 1(PfEMP1) is a family of variant surface antigens (VSA) that mediate the adhesion of parasite infected erythrocytes to capillary endothelial cells within host tissues. Opinion is divided over the role of PfEMP1 in the widespread endothelial activation associated with severe malaria. In a previous study we found evidence for differential associations between defined VSA subsets and specific syndromes of severe malaria: group A-like PfEMP1 expression and the "rosetting" phenotype were associated with impaired consciousness and respiratory distress, respectively. This study explores the involvement of widespread endothelial activation in these associations. METHODS: We used plasma angiopoietin-2 as a marker of widespread endothelial activation. Using logistic regression analysis, we explored the relationships between plasma angiopoietin-2 levels, parasite VSA expression and the two syndromes of severe malaria, impaired consciousness and respiratory distress. RESULTS: Plasma angiopoietin-2 was associated with both syndromes. The rosetting phenotype did not show an independent association with respiratory distress when adjusted for angiopoietin-2, consistent with a single pathogenic mechanism involving widespread endothelial activation. In contrast, group A-like PfEMP1 expression and angiopoietin-2 maintained independent associations with impaired consciousness when adjusted for each other. CONCLUSION: The results are consistent with multiple pathogenic mechanisms leading to severe malaria and heterogeneity in the pathophysiology of impaired consciousness. The observed association between group A-like PfEMP1 and impaired consciousness does not appear to involve widespread endothelial activation.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein 1(PfEMP1) is a family of variant surface antigens (VSA) that mediate the adhesion of parasite infected erythrocytes to capillary endothelial cells within host tissues. Opinion is divided over the role of PfEMP1 in the widespread endothelial activation associated with severe malaria. In a previous study we found evidence for differential associations between defined VSA subsets and specific syndromes of severe malaria: group A-like PfEMP1 expression and the "rosetting" phenotype were associated with impaired consciousness and respiratory distress, respectively. This study explores the involvement of widespread endothelial activation in these associations. METHODS: We used plasma angiopoietin-2 as a marker of widespread endothelial activation. Using logistic regression analysis, we explored the relationships between plasma angiopoietin-2 levels, parasite VSA expression and the two syndromes of severe malaria, impaired consciousness and respiratory distress. RESULTS: Plasma angiopoietin-2 was associated with both syndromes. The rosetting phenotype did not show an independent association with respiratory distress when adjusted for angiopoietin-2, consistent with a single pathogenic mechanism involving widespread endothelial activation. In contrast, group A-like PfEMP1 expression and angiopoietin-2 maintained independent associations with impaired consciousness when adjusted for each other. CONCLUSION: The results are consistent with multiple pathogenic mechanisms leading to severe malaria and heterogeneity in the pathophysiology of impaired consciousness. The observed association between group A-like PfEMP1 and impaired consciousness does not appear to involve widespread endothelial activation.

BACKGROUND: Although procurement consumes nearly 40% of Global Fund's money, no analyses have been published to show how costs vary across regions and time. This paper presents an analysis of malaria-related commodity procurement data from 79 countries, as reported through the Global Fund's price and quality reporting (PQR) system for the 2005-2012 period. METHODS: Data were analysed for the three most widely procured commodities for prevention, diagnosis and treatment of malaria. These were long-lasting insecticide-treated nets (LLINs), malaria rapid diagnostic tests (RDTs) and the artemether/lumefantrine (AL) combination treatment. Costs were compared across time (2005-2012), regions, and between individual procurement reported through the PQR and pooled procurement reported through the Global Fund's voluntary pooled procurement (VPP) system. All costs were adjusted for inflation and reported in US dollars. RESULTS: The data included 1,514 entries reported from 79 countries over seven years. Of these, 492 entries were for LLINs, 330 for RDTs and 692 for AL. Considerable variations were seen by commodity, although none showed an increase in cost. The costs for LLINs, RDTs and AL all dropped significantly over the period of analysis. Regional variations were also seen, with the cost for all three commodities showing significant variations. The median cost for a single LLIN ranged from USD 4.3 in East Asia to USD 5.0 in West and Central Africa. The cost of a single RDT was lowest in West and Central Africa at US$ 0.57, and highest in the Latin American region at US$ 1.1. AL had the narrowest margin of between US$ 0.06 per tablet in sub-Saharan Africa and South Asia, and US$ 0.08 in the Latin American and Eastern Europe regions. CONCLUSION: This paper concludes that global procurement costs do vary by region and have reduced overall over time. This suggests a mature market is operating when viewed from the global level, but regional variation needs further attention. Such analyses should be done more often to identify and correct market insufficiencies.

Early identification of causal genetic variants underlying antimalarial drug resistance could provide robust epidemiological tools for timely public health interventions. Using a novel natural genetics strategy for mapping novel candidate genes we analyzed >75,000 high quality single nucleotide polymorphisms selected from high-resolution whole-genome sequencing data in 27 isolates of Plasmodium falciparum. We identified genetic variants associated with susceptibility to dihydroartemisinin that implicate one region on chromosome 13, a candidate gene on chromosome 1 (PFA0220w, a UBP1 ortholog) and others (PFB0560w, PFB0630c, PFF0445w) with putative roles in protein homeostasis and stress response. There was a strong signal for positive selection on PFA0220w, but not the other candidate loci. Our results demonstrate the power of full-genome sequencing-based association studies for uncovering candidate genes that determine parasite sensitivity to artemisinins. Our study provides a unique reference for the interpretation of results from resistant infections.

Early identification of causal genetic variants underlying antimalarial drug resistance could provide robust epidemiological tools for timely public health interventions. Using a novel natural genetics strategy for mapping novel candidate genes we analyzed >75,000 high quality single nucleotide polymorphisms selected from high-resolution whole-genome sequencing data in 27 isolates of Plasmodium falciparum. We identified genetic variants associated with susceptibility to dihydroartemisinin that implicate one region on chromosome 13, a candidate gene on chromosome 1 (PFA0220w, a UBP1 ortholog) and others (PFB0560w, PFB0630c, PFF0445w) with putative roles in protein homeostasis and stress response. There was a strong signal for positive selection on PFA0220w, but not the other candidate loci. Our results demonstrate the power of full-genome sequencing-based association studies for uncovering candidate genes that determine parasite sensitivity to artemisinins. Our study provides a unique reference for the interpretation of results from resistant infections.

BACKGROUND: Nearly 40% of Global Fund money goes toward procurement. However, no analyses have been published to show how costs vary across regions and time, despite the availability of procurement data collected through the Global Fund's price and quality reporting system. METHODOLOGY: We analyzed data for the 3 most widely procured commodities for the prevention, diagnosis, and treatment of HIV. These were male condoms, HIV rapid tests, and the antiretroviral (ARV) combination of lamivudine/nevirapine/zidovudine. The compared costs, first across time (2005-2012), then across regions, and finally, between individual procurement reported through the price and quality reporting and pooled procurement reported through the Global Fund's voluntary pooled procurement system. All costs were adjusted for inflation and reported in US dollars. KEY FINDINGS: There were 2337 entries from 578 grants in 125 countries. The procurement cost for the ARV dropped substantially over the period, whereas those for condoms and HIV tests remained relatively stable. None of the commodity prices increased. Regional variations were pronounced for HIV tests, but minimal for condoms and the ARV. The unit cost for the 3-table ARV combination, for instance, varied between US$0.15 and US$0.23 in South Asia and the Eastern Europe/Central Asia regions, respectively, compared with a range of $0.23 (South Asia)-$1.50 (Eastern Europe/Central Asia) for a single diagnostic test. Pooled procurement lowered costs for condoms but not the other commodities. CONCLUSIONS: We showed how global procurement costs vary by region and time. Such analyses should be done more often to identify and correct market insufficiencies.

The Plasmodium falciparum genome is rich in regions of low amino acid complexity which evolve with few constraints on size. To explore the extent of diversity in these loci, we sequenced repeat regions in pfmdr1, pfmdr5, pfmdr6, pfmrp2, and the antigenic locus pfmsp8 in laboratory and cultured-adapted clinical isolates. We further assessed associations between the repeats and parasite in vitro responses to 7 antimalarials to determine possible adaptive roles of these repeats in drug tolerance. Our results show extensive repeat variations in the reference and clinical isolates in all loci. We also observed a modest increase in dihydroartemisinin activity in parasites harboring the pfmdr1 sequence profile 7-2-10 (reflecting the number of asparagine repeats, number of aspartate repeats, and number of asparagine repeats in the final series of the gene product) (P = 0.0321) and reduced sensitivity to chloroquine, mefloquine, quinine, and dihydroartemisinin in those with the 7-2-11 profile (P = 0.0051, 0.0068, 0.0011, and 0.0052, respectively). Interestingly, we noted an inverse association between two drugs whereby isolates with 6 asparagine repeats encoded by pfmdr6 were significantly more susceptible to piperaquine than those with 8 (P = 0.0057). Against lumefantrine, those with 8 repeats were, however, more sensitive (P = 0.0144). In pfmrp2, the 7-DNNNTS/NNNNTS (number of DNNNTS or NNNNTS motifs; underlining indicates dimorphism) repeat group was significantly associated with a higher lumefantrine 50% inhibitory concentration (IC50) (P = 0.008) than in those without. No associations were observed with pfmsp8. These results hint at the probable utility of some repeat conformations as markers of in vitro antimalarial response; hence, biochemical functional studies to ascertain their role in P. falciparum are required.

The Plasmodium falciparum genome is rich in regions of low amino acid complexity which evolve with few constraints on size. To explore the extent of diversity in these loci, we sequenced repeat regions in pfmdr1, pfmdr5, pfmdr6, pfmrp2, and the antigenic locus pfmsp8 in laboratory and cultured-adapted clinical isolates. We further assessed associations between the repeats and parasite in vitro responses to 7 antimalarials to determine possible adaptive roles of these repeats in drug tolerance. Our results show extensive repeat variations in the reference and clinical isolates in all loci. We also observed a modest increase in dihydroartemisinin activity in parasites harboring the pfmdr1 sequence profile 7-2-10 (reflecting the number of asparagine repeats, number of aspartate repeats, and number of asparagine repeats in the final series of the gene product) (P = 0.0321) and reduced sensitivity to chloroquine, mefloquine, quinine, and dihydroartemisinin in those with the 7-2-11 profile (P = 0.0051, 0.0068, 0.0011, and 0.0052, respectively). Interestingly, we noted an inverse association between two drugs whereby isolates with 6 asparagine repeats encoded by pfmdr6 were significantly more susceptible to piperaquine than those with 8 (P = 0.0057). Against lumefantrine, those with 8 repeats were, however, more sensitive (P = 0.0144). In pfmrp2, the 7-DNNNTS/NNNNTS (number of DNNNTS or NNNNTS motifs; underlining indicates dimorphism) repeat group was significantly associated with a higher lumefantrine 50% inhibitory concentration (IC50) (P = 0.008) than in those without. No associations were observed with pfmsp8. These results hint at the probable utility of some repeat conformations as markers of in vitro antimalarial response; hence, biochemical functional studies to ascertain their role in P. falciparum are required.

BACKGROUND: Children presenting to hospital with recent or current Plasmodium falciparum malaria are at increased the risk of invasive bacterial disease, largely enteric gram-negative organisms (ENGO), which is associated with increased mortality and recurrent morbidity. Although incompletely understood, the most likely source of EGNO is the bowel. We hypothesised that as a result of impaired gut-barrier function endotoxin (lipopolysaccharide), present in the cell-wall of EGNO and in substantial quantities in the gut, is translocated into the bloodstream, and contributes to the pathophysiology of children with severe malaria. METHODS: We conducted a prospective study in 257 children presenting with malaria to two hospitals in Kenya and Uganda. We analysed the clinical presentation, endotoxin and cytokine concentration. RESULTS: Endotoxaemia (endotoxin activity >/=0.4 EAA Units) was observed in 71 (27.6%) children but its presence was independent of both disease severity and outcome. Endotoxaemia was more frequent in children with severe anaemia but not specifically associated with other complications of malaria. Endotoxaemia was associated with a depressed inflammatory and anti-inflammatory cytokine response. Plasma endotoxin levels in severe malaria negatively correlated with IL6, IL10 and TGFbeta (Spearman rho: TNFalpha: r=-0.122, p=0.121; IL6: r=-0.330, p<0.0001; IL10: r=-0.461, p<0.0001; TGFbeta: r=-0.173, p<0.027). CONCLUSIONS: Endotoxaemia is common in malaria and results in temporary immune paralysis, similar to that observed in patients with sepsis and experimentally-induced endotoxaemia. Intense sequestration of P. falciparum-infected erythrocytes within the endothelial bed of the gut has been observed in pathological studies and may lead to gut-barrier dysfuction. The association of endotoxaemia with the anaemia phenotype implies that it may contribute to the dyserythropoesis accompanying malaria through inflammation. Both of these factors feasibly underpin the susceptibility to EGNO co-infection. Further research is required to investigate this initial finding, with a view to future treatment trials targeting mechanism and appropriate antimicrobial treatment.

BACKGROUND: Despite the publication of several studies on the subject, there is significant uncertainty regarding the burden of disease among adults in sub-Saharan Africa (sSA). OBJECTIVES: To describe the breadth of available data regarding causes of admission to hospital, to systematically analyze the methodological quality of these studies, and to provide recommendations for future research. DESIGN: We performed a systematic online and hand-based search for articles describing patterns of medical illnesses in patients admitted to hospitals in sSA between 1950 and 2010. Diseases were grouped into bodily systems using International Classification of Disease (ICD) guidelines. We compared the proportions of admissions and deaths by diagnostic category using chi2. RESULTS: Thirty articles, describing 86,307 admissions and 9,695 deaths, met the inclusion criteria. The leading causes of admission were infectious and parasitic diseases (19.8%, 95% confidence interval [CI] 19.6-20.1), respiratory (16.2%, 95% CI 16.0-16.5) and circulatory (11.3%, 95% CI 11.1-11.5) illnesses. The leading causes of death were infectious and parasitic (17.1%, 95% CI 16.4-17.9), circulatory (16%, 95% CI 15.3-16.8) and digestive (16.2%, 95% CI 15.4-16.9). Circulatory diseases increased from 3.9% of all admissions in 1950-59 to 19.9% in 2000-2010 (RR 5.1, 95% CI 4.5-5.8, test for trend p<0.00005). The most prevalent methodological deficiencies, present in two-thirds of studies, were failures to use standardized case definitions and ICD guidelines for classifying illnesses. CONCLUSIONS: Cardiovascular and infectious diseases are currently the leading causes of admissions and in-hospital deaths in sSA. Methodological deficiencies have limited the usefulness of previous studies in defining national patterns of disease in adults. As African countries pass through demographic and health transition, they need to significantly invest in clinical research capacity to provide an accurate description of the disease burden among adults for public health policy.