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Haemolytic disease of the newborn (HDN) is an immune haemolytic anaemia from maternal alloantibodies. Rh immunoglobulin (RhIg) prophylaxis can prevent alloimmunization to the D antigen. However, RhIg is not universally available in Uganda. ABO incompatibility also causes HDN. We determined the prevalence of HDN among newborn infants with jaundice in Uganda.We conducted a prospective cross-sectional study at Kawempe National Referral Hospital, Kampala, Uganda. Infants aged 0-14 days with neonatal jaundice (or total bilirubin50 μmol/L) were enrolled. Clinical evaluation and laboratory testing, including ABO, RhD typing and maternal antibody screen, were performed.A total of 466 babies were enrolled. The mean (SD) age was 3.4 (1.5) days. Of newborn babies with jaundice, 17.2% (80/466) had HDN. Babies with HDN had lower haemoglobin (SD); 15.7 (2.7) compared with those without HDN; 16.4 (2.4) g/dL, p = 0.016; and a higher bilirubin (interquartile range); 241 (200-318) compared with those without HDN; 219 (191-263) μmol/L, p 0.001. One baby had anti-D HDN, while 46/466 had HDN from an ABO incompatibility (anti-A 43.5% and anti-B 56.5%); 82% of babies with HDN also had suspected neonatal sepsis or birth asphyxia. About 79.2% (57/72) of mothers did not have ABO/Rh blood group performed antenatally. All infants with HDN survived except one.Among newborn infants with jaundice, HDN is not rare. The majority is due to ABO HDN affecting group A and group B babies equally. Ensuring routine ABO/Rh grouping for all pregnant women is an area for improvement.

Background Children hospitalised with severe anaemia in malaria-endemic areas are at a high risk of dying or being readmitted within six months of discharge. A trial in Kenya and Uganda showed that three months of postdischarge malaria chemoprevention (PDMC) with monthly dihydroartemisinin-piperaquine (DP) substantially reduced this risk. The World Health Organization recently included PDMC in its malaria chemoprevention guidelines. We conducted a cost-effectiveness analysis of community-based PDMC delivery (supplying all three PDMC-DP courses to caregivers at discharge to administer at home), facility-based PDMC delivery (monthly dispensing of PDMC-DP at the hospital), and the standard of care (no PDMC). Methods We combined data from two recently completed trials; one placebo-controlled trial in Kenya and Uganda collecting efficacy data (May 6, 2016 until November 15, 2018; n=1049), and one delivery mechanism trial from Malawi collecting adherence data (March 24, 2016 until October 3, 2018; n=375). Cost data were collected alongside both trials. Three Markov decision models, one each for Malawi, Kenya, and Uganda, were used to compute incremental cost-effectiveness ratios expressed as costs per quality-adjusted life-year (QALY) gained. Deterministic and probabilistic sensitivity analyses were performed to account for uncertainty. Findings Both PDMC strategies were cost-saving in each country, meaning less costly and more effective in increasing health-adjusted life expectancy than the standard of care. The estimated incremental cost savings for community-based PDMC compared to the standard of care were US$ 22·10 (Malawi), 38·52 (Kenya), and 26·23 (Uganda) per child treated. The incremental effectiveness gain using either PDMC strategy varied between 0·3 and 0·4 QALYs. Community-based PDMC was less costly and more effective than facility-based PDMC. These results remained robust in sensitivity analyses. Interpretation PDMC under implementation conditions is cost-saving. Caregivers receiving PDMC at discharge is a cost-effective delivery strategy for implementation in malaria-endemic southeastern African settings. publishedVersion

Children recovering from severe malarial anaemia (SMA) remain at high risk of readmission and death after discharge from hospital. However, a recent trial found that post-discharge malaria chemoprevention (PDMC) reduces this risk. We developed a mathematical model describing the daily incidence of uncomplicated and severe malaria requiring readmission among 0-5 year-old children after hospitalised SMA. We fitted the model to a multicentre clinical PDMC trial using Bayesian methods. We then modelled the potential impact of PDMC across malaria-endemic African countries. In the 19 highest-burden countries, we estimate that only 2-5 children need to be given PDMC to prevent one hospitalised malaria episode, and less than 100 to prevent one death. If all hospitalised SMA cases access PDMC, 37,000 (range 16,000-84,000) malaria-associated readmissions could be prevented annually, depending on access to hospital care. We estimate that recurrent SMA post-discharge constitutes 18-29% of all SMA episodes in higher transmission settings.

BACKGROUND Children who have been hospitalized with severe anemia in areas of Africa in which malaria is endemic have a high risk of readmission and death within 6 months after discharge. No prevention strategy specifically addresses this period. METHODS We conducted a multicenter, two-group, randomized, placebo-controlled trial in nine hospitals in Kenya and Uganda to determine whether 3 months of malaria chemoprevention could reduce morbidity and mortality after hospital discharge in children younger than 5 years of age who had been admitted with severe anemia. All children received standard in-hospital care for severe anemia and a 3-day course of artemether–lumefantrine at discharge. Two weeks after discharge, children were randomly assigned to receive dihydroartemisinin–piperaquine (chemoprevention group) or placebo, administered as 3-day courses at 2, 6, and 10 weeks after discharge. Children were followed for 26 weeks after discharge. The primary outcome was one or more hospital readmissions for any reason or death from the time of randomization to 6 months after discharge. Conditional risk-set modeling for recurrent events was used to calculate hazard ratios with the use of the Prentice–Williams–Peterson total-time approach. RESULTS From May 2016 through May 2018, a total of 1049 children underwent randomization; 524 were assigned to the chemoprevention group and 525 to the placebo group. From week 3 through week 26, a total of 184 events of readmission or death occurred in the chemoprevention group and 316 occurred in the placebo group (hazard ratio, 0.65; 95% confidence interval [CI], 0.54 to 0.78; P

Background: The majority of blood transfusion safety strategies recommended by the WHO for resource-poor countries focus mainly on reducing the risk of transfusion-transmitted infections (TTIs). Other technologies such as leucocyte reduc- tion may represent complementary strategies for improving transfusion safety. Objective: To evaluate the role of using leucocyte reduced blood in a resource-poor country. Methods: Pre-storage leucocyte reduced (LR) red blood cells (RBCs) were specially prepared for the Tissue Oxygenation by Transfusion in severe Anaemia and Lactic acidosis (TOTAL) study, at the Uganda Blood Transfusion Services from Feb- ruary 2013 through May 2015. Quality control tests were performed to evaluate the procedure, and the incremental cost of an LR–RBC unit was estimated. Results: A total of 608 RBCs units were leucocyte reduced. Quality control tests were performed on 55 random RBCs units. The median (IQR) residual leucocyte count was 4 (0•5-10) WBC/uL, equivalent to 1•8x106 WBC per unit. The estimated incremental unit cost of leucocyte reduction was $37 USD per LR RBC unit. Conclusion: Leucocyte reduction of blood in a resource-poor country is doable although relatively costly. As such, its value in resource-poor countries should be weighed against other transfusion safety propositions. Keywords: Blood transfusion safety; leucocyte reduction; resource-poor countries.

Background In resource-poor settings, transfused children often experience recurrence of severe anemia (SA) following discharge from hospital. This study determined the factors associated with recurrent severe anemia (RSA) among previously transfused Ugandan children aged less than 5 years. Methods A case-control study was conducted in five hospitals in Uganda from March 2017 to September 2018. We prospectively enrolled 196 hospitalised children who had been transfused for severe anemia 2 weeks to 6 months prior to enrollment. Of these, 101 children (cases) were re-admitted with a hemoglobin [Hb] level of ≤6 g/dL and required transfusion; and 95 children (age-matched controls) were admitted for other clinical illness with a Hb > 6 g/dL. Children known to have sickle cell anemia, cancer, or bleeding disorders were excluded. Clinical and laboratory evaluation were done. Conditional logistic regression adjusted for age, was used to determine factors associated with RSA. Results The median time (IQR) between the earlier transfusion and enrollment was 3.5 (1.9–5.7) months for cases, and was 5.0 (2.9–6.0) months for controls (p-value = 0.015). Risk factors (adjusted odds ratio, 95% confidence interval, and significance) for development of RSA were: hemoglobinuria (36.33, 2.19–600.66, p = 0.012); sickle cell anemia – newly diagnosed (20.26, 2.33–176.37, p = 0.006); history of earlier previous transfusions (6.95, 1.36–35.61, p = 0.020) and malaria infection (6.47, 1.17–35.70, p = 0.032). Conclusion Malaria chemoprevention, follow up visit for Hb check after discharge from hospital and sickle cell screening among previously transfused children represent practical strategies to prevent and identify children at risk for recurrent severe anemia. The cause of hemoglobinuria in children merits further investigations.

Objective To describe local perceptions of blood transfusion for children with severe anaemia in Uganda. Background Blood transfusion is a common emergency treatment for children with severe anaemia and saves millions of lives of African children. However, the perceptions of transfusion recipients have not been well studied. A better understanding of the perceived risk may improve transfusion care. Methods A qualitative study based on 16 in-depth interviews of caregivers of transfused children, and six focus group discussions with community members was conducted in three regions of Uganda between October and November 2017. Results Caregivers of children and community members held blood transfusion in high regard and valued it as life-saving. However, there were widespread perceived transfusion risks, including: Human immunodeficiency virus (HIV) transmission, too rapid blood infusion and blood incompatibility. Other concerns were: fatality, changes in behaviour, donor blood being 'too strong' and use of animal blood. In contrast, recent transfusion, older age, knowledge of HIV screening of blood for transfusion, faith in God and having a critically ill child were associated with less fear about transfusion. Respondents also emphasised challenges to transfusion services access including distance to hospitals, scarcity of blood and health workers' attitudes. Conclusion Perceptions of the community and caregivers of transfused children in Uganda about blood transfusion were complex: transfusion is considered life-saving but there were strong perceived transfusion risks of HIV transmission and blood incompatibility. Addressing community perceptions and facilitating access to blood transfusion represent important strategies to improve paediatric transfusion care.

Background Children hospitalised with severe anaemia in malaria endemic areas in Africa are at high risk of readmission or death within 6 months post-discharge. Currently, no strategy specifically addresses this period. In Malawi, 3 months of post-discharge malaria chemoprevention (PMC) with monthly treatment courses of artemether-lumefantrine given at discharge and at 1 and 2 months prevented 30% of all-cause readmissions by 6 months post-discharge. Another efficacy trial is needed before a policy of malaria chemoprevention can be considered for the post-discharge management of severe anaemia in children under 5 years of age living in malaria endemic areas. Objective We aim to determine if 3 months of PMC with monthly 3-day treatment courses of dihydroartemisinin-piperaquine is safe and superior to a single 3-day treatment course with artemether-lumefantrine provided as part of standard in-hospital care in reducing all-cause readmissions and deaths (composite primary endpoint) by 6 months in the post-discharge management of children less than 5 years of age admitted with severe anaemia of any or undetermined cause. Methods/design This is a multi-centre, two-arm, placebo-controlled, individually randomised trial in children under 5 years of age recently discharged following management for severe anaemia. Children in both arms will receive standard in-hospital care for severe anaemia and a 3-day course of artemether-lumefantrine at discharge. At 2 weeks after discharge, surviving children will be randomised to receive either 3-day courses of dihydroartemisinin-piperaquine at 2, 6 and 10 weeks or an identical placebo and followed for 26 weeks through passive case detection. The trial will be conducted in hospitals in malaria endemic areas in Kenya and Uganda. The study is designed to detect a 25% reduction in the incidence of all-cause readmissions or death (composite primary outcome) from 1152 to 864 per 1000 child years (power 80%, α = 0.05) and requires 520 children per arm (1040 total children). Results Participant recruitment started in May 2016 and is ongoing. Trial registration ClinicalTrials.gov, NCT02671175. Registered on 28 January 2016. Electronic supplementary material The online version of this article (10.1186/s13063-018-2972-1) contains supplementary material, which is available to authorized users.

Importance Severe anemia, defined as a hemoglobin level of less than 5.0 g/dL, affects millions of children worldwide. The brain has a high basal demand for oxygen and is especially vulnerable to hypoxemia. Previous studies have documented neurocognitive impairment in children with severe anemia. Data on cerebral tissue oxygenation in children with severe anemia and their response to blood transfusion are limited. Objective To measure hemoglobin saturation in cerebral tissue (cerebral tissue oxygen saturation [tSo 2 ]) before, during, and after blood transfusion in a cohort of children presenting to hospital with severe anemia. Design, Setting, and Participants This was a prospective, observational cohort study conducted from February 2013 through May 2015 and analyzed in July 2015 at a university hospital pediatric acute care facility in Kampala, Uganda, of 128 children, ages 6 to 60 months who were enrolled in a larger clinical trial, with a presenting hemoglobin level of less than 5.0 g/dL and a blood lactate level greater than 5mM. Most children had either malaria or sickle cell disease. Exposures Red blood cell (RBC) transfusion given as 10 mL/kg over 120 minutes. Main Outcomes and Measures Clinical and laboratory characteristics of children with pretransfusion cerebral tSo 2 levels less than 65%, 65% to 75%, and greater than 75%. Change in cerebral tSo 2 as a result of transfusion. Results Of 128 children included in the study, oximetry results in 8 cases were excluded owing to motion artifacts; thus, 120 were included in this analysis. Cerebral tSo 2 values prior to transfusion ranged from 34% to 87% (median, 72%; interquartile range [IQR], 65%-76%). Eighty-one children (67%) demonstrated an initial cerebral tSo 2 level (≤75%) corresponding to an oxygen extraction ratio greater than 0.36. Patients with sickle cell disease (n = 17) and malaria (n = 15) contributed in nearly equal numbers to the subgroup with an initial cerebral tSo 2 ( 2 level were poor predictors of cerebral oxygen saturation. Following RBC transfusion, the median (IQR) cerebral tSo 2 level increased to 78% (73%-82%) ( P 2 level greater than 75%. Conclusions and Relevance Severe anemia in children is frequently associated with low cerebral oxygenation levels as measured by near-infrared spectroscopy. Hemoglobin level and lactate concentration did not predict low cerebral tSo 2 levels. Cerebral tSo 2 levels increase with RBC transfusion with different patterns of response. More studies are needed to evaluate the use of noninvasive cerebral tissue oximetry in the care of children with severe anemia.

Summary As a leading cause of childhood mortality worldwide, selection pressure by Plasmodium falciparum continues to shape the human genome. Severe disturbances within the microcirculation result from the adhesion of infected erythrocytes to host receptors on monocytes, platelets, and endothelium. In this prospective study, we compared expression of all major host cytoadhesion receptors among Ugandan children presenting with uncomplicated malaria (n = 1078) versus children with severe malaria (n = 855), including cerebral malaria (n = 174), severe anaemia (n = 522), and lactic acidosis (n = 154). We report a significant survival advantage attributed to blood group O and increased monocyte expression of CD36 and ICAM1 (CD54). The high case fatality rate syndromes of cerebral malaria and lactic acidosis were associated with high platelet CD36 expression and thrombocytopenia, and severe malaria anaemia was characterized by low ICAM1 expression. In a logistic regression model of disease severity, odds ratios for the mitigating effects of blood group O, CD36, and ICAM1 phenotypes were greater than that of sickle haemoglobin. Host genetic adaptations to Plasmodium falciparum suggest new potential malaria treatment strategies.

Although millions of transfusions are given annually worldwide, the effect of red blood cell (RBC) unit storage duration on oxygen delivery is uncertain.To determine if longer-storage RBC units are not inferior to shorter-storage RBC units for tissue oxygenation as measured by reduction in blood lactate levels and improvement in cerebral tissue oxygen saturation among children with severe anemia.Randomized noninferiority trial of 290 children (aged 6-60 months), most with malaria or sickle cell disease, presenting February 2013 through May 2015 to a university-affiliated national referral hospital in Kampala, Uganda, with a hemoglobin level of 5 g/dL or lower and a lactate level of 5 mmol/L or higher.Patients were randomly assigned to receive RBC units stored 25 to 35 days (longer-storage group; n = 145) vs 1 to 10 days (shorter-storage group; n = 145). All units were leukoreduced prior to storage. All patients received 10 mL/kg of RBCs during hours 0 through 2 and, if indicated per protocol, an additional 10 mL/kg during hours 4 through 6.The primary outcome was the proportion of patients with a lactate level of 3 mmol/L or lower at 8 hours using a margin of noninferiority equal to an absolute difference of 25%. Secondary measures included noninvasive cerebral tissue oxygen saturation during the first transfusion, clinical and laboratory changes up to 24 hours, and survival and health at 30 days after transfusion. Adverse events were monitored up to 24 hours.In the total population of 290 children, the mean (SD) presenting hemoglobin level was 3.7 g/dL (1.3) and mean lactate level was 9.3 mmol/L (3.4). Median (interquartile range) RBC unit storage was 8 days (7-9) for shorter storage vs 32 days (30-34) for longer storage without overlap. The proportion achieving the primary end point was 0.61 (95% CI, 0.52 to 0.69) in the longer-storage group vs 0.58 (95% CI, 0.49 to 0.66) in the shorter-storage group (between-group difference, 0.03 [95% CI, -0.07 to ∞], P .001), meeting the prespecified margin of noninferiority. Mean lactate levels were not statistically different between the 2 groups at 0, 2, 4, 6, 8, or 24 hours. Kaplan-Meier analysis and global nonlinear regression revealed no statistical difference in lactate reduction between the 2 groups. Clinical assessment, cerebral oxygen saturation, electrolyte abnormalities, adverse events, survival, and 30-day recovery were also not significantly different between the groups.Among children with lactic acidosis due to severe anemia, transfusion of longer-storage compared with shorter-storage RBC units did not result in inferior reduction of elevated blood lactate levels. These findings have relevance regarding the efficacy of stored RBC transfusion for patients with critical tissue hypoxia and lactic acidosis due to anemia.clinicaltrials.gov Identifier: NCT01586923.

Cerebral malaria (CM) is associated with excessive inflammatory responses and endothelial activation. Sphingosine 1-phosphate (S1P) is a signaling sphingolipid implicated in regulating vascular integrity, inflammation and T-cell migration. We hypothesized that altered S1P signaling during malaria contributes to endothelial activation and inflammation, and show that plasma S1P levels were decreased in Ugandan children with CM compared with children with uncomplicated malaria. Using the Plasmodium berghei ANKA (PbA) model of experimental CM (ECM), we demonstrate that humanized S1P lyase (hS1PL)(-/-) mice with reduced S1P lyase activity (resulting in increased bio-available S1P) had improved survival compared with wild-type littermates. Prophylactic and therapeutic treatment of infected mice with compounds that modulate the S1P pathway and are in human trials for other conditions (FTY720 or LX2931) significantly improved survival in ECM. FTY720 treatment improved vascular integrity as indicated by reduced levels of soluble intercellular adhesion molecule (sICAM), increased angiopoietin 1 (Ang1) (regulator of endothelial quiescence) levels, and decreased Evans blue dye leakage into brain parenchyma. Furthermore, treatment with FTY720 decreased IFNγ levels in plasma as well as CD4(+) and CD8(+) T-cell infiltration into the brain. Finally, when administered during infection in combination with artesunate, FTY720 treatment resulted in increased survival to ECM. These findings implicate dysregulation of the S1P pathway in the pathogenesis of human and murine CM and suggest a novel therapeutic strategy to improve clinical outcome in severe malaria.

To investigate whether hand-carried ultrasound technology may be valuable in the assessment of children with acute malaria. Every year, approximately 800,000 children under the age of 5 yrs die of complications of Plasmodium falciparum malaria infection. The advent of hand-carried ultrasound technology has made diagnostic ultrasonography possible in underresourced settings.We performed a pilot observational study collecting clinical data and performing ultrasound examinations on children diagnosed with P. falciparum malaria infection. The targeted ultrasound examination included measurement of optic nerve sheath diameter, color transcranial Doppler insonation of the cerebral vasculature, cardiac ultrasound, and abdominal ultrasound.Pediatric acute care unit of Mulago Hospital in Kampala, Uganda.Thirty-three hospitalized children between the ages of 6 months and 12 yrs with documented acute P. falciparum infection.Targeted bedside ultrasound examination.Increased optic nerve sheath diameter was observed in one third of all patients with malaria and in 100% of the patients diagnosed with cerebral malaria. Although higher-than-normal cerebral blood flow velocities were demonstrated in three (25%) of 12 patients with severe anemia, most patients demonstrated a normal cerebral blood flow velocity, suggesting a blunted response to anemia. We did not find evidence of pulmonary hypertension by cardiac ultrasound, and cardiac function did not seem depressed, even among patients with severe anemia and lactic acidosis. Finally, spleen size as determined by palpation significantly overestimated the true incidence of splenomegaly as measured by ultrasound (48% and 24%, respectively).A targeted ultrasound examination focusing on optic nerve sheath diameter, color transcranial Doppler, cardiac ultrasound, and spleen size may prove useful for patient classification, risk stratification, research studies, and treatment monitoring in pediatric malaria. More studies should be done.

Prior studies have suggested that transfusion of stored red blood cells (RBCs) with increased levels of cell-free hemoglobin might reduce the bioavailability of recipient nitric oxide (NO) and cause myocardial strain.Ugandan children (ages 6-60 months) with severe anemia and lactic acidosis were randomly assigned to receive RBCs stored 1-10 days versus 25-35 days. B-type natriuretic peptide (BNP), vital signs, renal function test results, and plasma hemoglobin were measured. Most children had either malaria or sickle cell disease and were thus at risk for reduced NO bioavailability.Seventy patients received RBCs stored 1-10 days, and 77 received RBCs stored 25-35 days. The median (interquartile range) cell-free hemoglobin was nearly 3 times higher in longer-storage RBCs (26.4 [15.5-43.4] μmol/L) than in shorter-storage RBCs (10.8 [7.8-18.6] μmol/L), P.0001. Median (interquartile range) BNP 2 hours posttransfusion was 156 (59-650) pg/mL (shorter storage) versus 158 (59-425) pg/mL (longer storage), P = .76. BNP values 22 hours posttransfusion were 110 (46-337) pg/mL (shorter storage) versus 96 (49-310) pg/mL (longer storage), P = .76. Changes in BNP within individuals from pretransfusion to 2 hours (or 22 hours) posttransfusion were not significantly different between the study groups. BNP change following transfusion did not correlate with the concentration of cell-free hemoglobin in the RBC supernatant. Blood pressure, blood urea nitrogen, creatinine, and change in plasma hemoglobin were not significantly different in the 2 groups.In a randomized trial among children at risk for reduced NO bioavailability, we found that BNP, blood pressure, creatinine, and plasma hemoglobin were not higher in patients receiving RBCs stored for 25-35 versus 1-10 days.

Background Severe and fatal malaria are associated with dysregulated host inflammatory responses to infection. Chitinase 3-like 1 (CHI3L1) is a secreted glycoprotein implicated in regulating immune responses. Expression and function of CHI3L1 in malaria infection were investigated. Methods Plasma levels of CHI3L1 were quantified in a case–control study of Ugandan children presenting with Plasmodium falciparum malaria. CHI3L1 levels were compared in children with uncomplicated malaria (UM; n = 53), severe malarial anaemia (SMA; n = 59) and cerebral malaria (CM; n = 44) using the Kruskall Wallis-test, and evaluated for utility in predicting fatal (n = 23) versus non-fatal (n = 80) outcomes in severe disease using the Mann Whitney U test, receiver operating characteristic curves, and combinatorial analysis. Co-culture of P. falciparum with human peripheral blood mononuclear cells and the Plasmodium berghei ANKA experimental model of cerebral malaria were used to examine the role of CHI3L1 in severe malaria. Results In children presenting with falciparum malaria, CHI3L1 levels were increased in SMA and CM versus UM (p

The host immune response plays an important role in the onset and progression of cerebral malaria (CM). The complement system is an essential component of the innate immune response to malaria, and its activation generates the anaphylatoxin C5a. To test the hypothesis that C5a signaling contributes to the pathogenesis of CM, we investigated a causal role for the C5a receptors C5aR and C5L2 in a mouse model of experimental CM (ECM) induced by Plasmodium berghei ANKA infection, and using a case-control design, we examined levels of C5a in plasma samples from Ugandan children presenting with CM or uncomplicated malaria (UM). In the ECM model, C5aR −/− mice displayed significantly improved survival compared to their wild-type (WT) counterparts ( P = 0.004), whereas C5L2 −/− mice showed no difference in survival from WT mice. Improved survival in C5aR −/− mice was associated with reduced levels of the proinflammatory cytokines tumor necrosis factor (TNF) and gamma interferon (IFN-γ) and the chemokine, monocyte chemoattractant protein 1 (MCP-1) (CCL2). Furthermore, endothelial integrity was enhanced, as demonstrated by increased levels of angiopoietin-1, decreased levels of angiopoietin-2 and soluble ICAM-1, and decreased Evans blue extravasation into brain parenchyma. In the case-control study, the median levels of C5a at presentation were significantly higher in children with CM versus those in children with UM (43.7 versus 22.4 ng/ml; P < 0.001). These findings demonstrate that C5a is dysregulated in human CM and contributes to the pathogenesis of ECM via C5aR-dependent inflammation and endothelial dysfunction.

Malaria remains a challenging diagnosis with variable clinical presentation and a wide spectrum of disease severity. Using a structured case report form, we prospectively assessed 1,933 children at Mulago Hospital in Kampala, Uganda with acute Plasmodium falciparum malaria. Children with uncomplicated malaria significantly differed from those with severe disease for 17 features. Among 855 children with severe disease, the case-fatality rate increased as the number of severity features increased. Logistic regression identified five factors independently associated with death: cerebral malaria, hypoxia, severe thrombocytopenia, leukocytosis, and lactic acidosis. Cluster analysis identified two groups: one combining anemia, splenomegaly, and leukocytosis; and a second group centered on death, severe thrombocytopenia, and lactic acidosis, which included cerebral malaria, hypoxia, hypoglycemia, and hyper-parasitemia. Our report updates previous clinical descriptions of severe malaria, quantifies significant clinical and laboratory inter-relationships, and will assist clinicians treating malaria and those planning or assessing future research ({"type":"clinical-trial","attrs":{"text":"NCT00707200","term_id":"NCT00707200"}}NCT00707200) (www.clinicaltrials.gov).

Background Severe falciparum malaria (SM) pathogenesis has been attributed, in part, to deleterious systemic host inflammatory responses to infection. High mobility group box 1 (HMGB1) protein is an important mediator of inflammation implicated in sepsis pathophysiology. Methods Plasma levels of HMGB1 were quantified in a cohort of febrile Ugandan children with Plasmodium falciparum infection, enrolled in a prospective observational case-controlled study, using a commercial enzyme-linked immunosorbent assay. The utility of HMGB1 to distinguish severe malaria (SM; n = 70) from uncomplicated malaria (UM; n = 33) patients and fatal (n = 21) versus non-fatal (n = 82) malaria, at presentation, was examined. Receiver operating characteristic curve analysis was used to assess the prognostic accuracy of HMGB1. The ability of P. falciparum-parasitized erythrocytes to induce HMGB1 from peripheral blood mononuclear cells was assessed in vitro. The effect of an anti-HMGB1 neutralizing antibody on disease outcome was assessed in the experimental Plasmodium berghei ANKA rodent parasite model of SM. Mortality and parasitaemia was assessed daily and compared to isotype antibody-treated controls. Results Elevated plasma HMGB1 levels at presentation were significantly associated with SM and a subsequent fatal outcome in paediatric patients with P. falciparum infection. In vitro, parasitized erythrocytes induced HMGB1 release from human peripheral blood mononuclear cells. Antibody-mediated neutralization of HMGB1 in the experimental murine model of severe malaria failed to reduce mortality. Conclusion These data suggest that elevated HMGB1 is an informative prognostic marker of disease severity in human SM, but do not support HMGB1 as a viable target for therapeutic intervention in experimental murine SM.

Each year approximately 100 million units of blood are transfused worldwide. Current regulations allow RBCs to be stored in approved solutions for up to five or six weeks. Regulatory agencies use cell survival studies and in vitro markers of hemolysis, rather than measures of oxygen delivery, to establish the maximal duration of blood storage. During storage, RBCs undergo cumulative structural, biochemical, and enzymatic changes that collectively might impair the ability of erythrocytes to deliver oxygen to tissues and contribute to adverse patient outcomes. Study design and Methods: We conducted a prospective, randomized, controlled, clinical trial comparing short-storage versus longer-storage RBCs for tissue oxygen delivery as measured by reduction in blood lactate levels in severe anemia. We studied 290 patients presenting to a university hospital urgent care facility with a hemoglobin ≤5g/dL and lactate ≥5mM. Patients were children, age 6 to 60 months, whose lactic acidosis was due to severe anemia and who did not have shock, trauma, impaired cardiac function, refractory hypoxia, need for pressors, liver disease or tissue injury. Subjects were randomly assigned to receive leukoreduced RBCs stored 1-10 days (n=145) versus 25-35 days (n=145). All patients received 10mL/kg of RBCs during hours 0-2; and, if indicated per protocol, an additional 10mL/kg during hours 4-6. The study was conducted in Kampala, Uganda and registered as #NCT01586923 at clinicaltrials.gov. Results: Hour 0 was the start of transfusion.We measured blood lactate levels at time 0, 2, 4, 6, 8 and 24 hours following transfusion. The primary outcome was the proportion of patients achieving a lactate ≤3mM at 8 hours. Other measures included cerebral tissue oxygen saturation measured non-invasively, clinical and laboratory changes following transfusion, survival, and 30-day follow-up. At presentation, the mean hemoglobin was 3.7 ±1.3g/dL and mean lactate was 9.3 ±3.4mM. RBC storage averaged 7.8 ±2.1 days in the shorter-storage arm and 31.6 ±2.8 days in the longer-storage arm without overlap. See Fig 1A. The proportion achieving a lactate ≤3mM at 8 hours was 0.58 (95% CI, 0.49-0.66, shorter-storage) versus 0.61 (95% CI, 0.52-0.69, longer-storage), p=0.72, a result meeting the pre-specified margin of non-inferiority. Mean lactate levels were not statistically different between the two arms at 2, 4, 6, 8 or 24 hours. See Fig 1B. Kaplan-Meier analysis and global non-linear regression revealed no difference in lactate clearance between the shorter-storage and longer-storage groups. Clinical assessment, serial measurements of hemoglobin concentration, cerebral tissue oxygen saturation, and electrolyte abnormalities improved to the same degree in the two groups following transfusion. Adverse events, survival, and 30-day recovery were not significantly different between the two groups. Pre-specified sub-group analysis among patients receiving 20mL/kg of RBCs revealed no significant differences in outcomes. Given the severity of anemia, transfusions represented 60% to 90% of the patients' pre-transfusion red cell mass. Conclusion and Relevance: This is the first major randomized trial specifically testing whether prolonged storage RBCs deliver oxygen as well as short-storage RBCs. We tested the two extremes of blood storage duration and found that longer-storage RBCs were not inferior to shorter-storage RBCs for oxygen delivery as measured by reduction of elevated blood lactate levels, improved cerebral oxygen saturation, clinical outcomes, laboratory results, or adverse events. The results carry relevance for health policy decisions regarding the acceptable duration of RBC storage worldwide. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Most studies on HIV serostatus disclosure and adolescents focus on whether, how and when to disclose to adolescents their HIV diagnosis. Fewer studies have examined HIV serostatus disclosure by adolescents who know they are infected with HIV. This study presents qualitative data examining HIV serostatus and treatment disclosure practices and concerns of young people living with HIV in Uganda and the extent to which they are satisfied with current norms around HIV serostatus and treatment disclosure. We conducted two focus groups and interviewed 20 HIV-infected young people aged 15-23 receiving HIV care and treatment at the Transition Clinic in Kampala. Respondents perceived disclosure as a relationship encompassing both communication and self-conduct. Adolescents employed unique strategies to disclose their HIV status, notably joking to "test the waters" and emotionally prepare the other person before later disclosing in a more serious manner. Findings reinforce the idea that HIV disclosure is a process, not a one-time event. Interviewees anticipated both positive and negative outcomes of disclosure, including financial and emotional support, stigma, discrimination and rejection. They described a sense of violation of their autonomy when confidentiality was breached by third party disclosure, and also expressed fear of emotional distress for their loved ones. Although adolescents yearned to be in control of information about their HIV status and treatment, they have little space to call their own, and privacy is often compromised, especially because in traditional African settings, young people are considered to be dependents under the full responsibility of caregivers. Further exploration of disclosure outcomes and strategies specific to adolescents can help better tailor interventions towards youth. Antiretroviral therapy programmes should consider counselling for caretakers to appreciate and respect the privacy and disclosure concerns of their HIV-infected children.

Background Severe malaria is a leading cause of childhood mortality in Africa. However, at presentation, it is difficult to predict which children with severe malaria are at greatest risk of death. Dysregulated host inflammatory responses and endothelial activation play central roles in severe malaria pathogenesis. We hypothesized that biomarkers of these processes would accurately predict outcome among children with severe malaria. Methodology/Findings Plasma was obtained from children with uncomplicated malaria (n = 53), cerebral malaria (n = 44) and severe malarial anemia (n = 59) at time of presentation to hospital in Kampala, Uganda. Levels of angiopoietin-2, von Willebrand Factor (vWF), vWF propeptide, soluble P-selectin, soluble intercellular adhesion molecule-1 (ICAM-1), soluble endoglin, soluble FMS-like tyrosine kinase-1 (Flt-1), soluble Tie-2, C-reactive protein, procalcitonin, 10 kDa interferon gamma-induced protein (IP-10), and soluble triggering receptor expressed on myeloid cells-1 (TREM-1) were determined by ELISA. Receiver operating characteristic (ROC) curve analysis was used to assess predictive accuracy of individual biomarkers. Six biomarkers (angiopoietin-2, soluble ICAM-1, soluble Flt-1, procalcitonin, IP-10, soluble TREM-1) discriminated well between children who survived severe malaria infection and those who subsequently died (area under ROC curve>0.7). Combinational approaches were applied in an attempt to improve accuracy. A biomarker score was developed based on dichotomization and summation of the six biomarkers, resulting in 95.7% (95% CI: 78.1–99.9) sensitivity and 88.8% (79.7–94.7) specificity for predicting death. Similar predictive accuracy was achieved with models comprised of 3 biomarkers. Classification tree analysis generated a 3-marker model with 100% sensitivity and 92.5% specificity (cross-validated misclassification rate: 15.4%, standard error 4.9%). Conclusions We identified novel host biomarkers of pediatric severe and fatal malaria (soluble TREM-1 and soluble Flt-1) and generated simple biomarker combinations that accurately predicted death in an African pediatric population. While requiring validation in further studies, these results suggest the utility of combinatorial biomarker strategies as prognostic tests for severe malaria.

Abstract 666 Background: Plasmodium falciparum malaria has been called the greatest force in human evolution as a result of its disproportionate lethality among children and its longstanding presence in human history. Malaria's complex pathogenesis is based in part on cytoadhesion, which is mediated by a parasite-derived surface protein (PfEMP1) expressed on infected erythrocytes. PfEMP1 binds with ligands on red cells, platelets, and endothelium, and through the lectin-like DBL1α domain, binds preferentially to blood Group A. This contributes to the obstruction of microvascular perfusion and to cerebral malaria, lactic acidosis, and death. Initial studies demonstrated increased cytoadhesion to Group A RBC in vitro, suggesting that Group A hosts may be at risk of greater morbidity with P falciparum. Subsequently, retrospective clinical studies demonstrated a higher proportion of Group A individuals among those with severe disease. Recently, attention has focused on malaria pathogenesis and von Willebrand factor, which is elevated in Group A individuals. While the cumulative evidence suggests a central role for ABO in malaria, proof for a driving effect on the evolutionary distribution of ABO blood groups has awaited definitive study of the relationship between ABO and P falciparum malaria mortality. We report here initial findings from the Cytoadherence in Pediatric Malaria (CPM) Study (clinicaltrials.gov, NCT 00707200) which was designed to examine associations between malaria outcomes and host blood groups implicated in erythrocyte cytoadhesion. Methods: We performed a 2 year prospective observational study of children with acute malaria (age 6 mo – 12 yr) presenting for care at Mulago Hospital, Uganda. HIV positive patients were excluded from analysis. We enrolled children who met pre-study WHO criteria for either Uncomplicated Malaria (UM) or Severe Malaria (SM). Patients were followed from the time of presentation to discharge or death. ABO blood groups were determined by standard hemagglutination. The study was approved by the Uganda National Council on Science and Technology and all participating institutions. Results: At the study's conclusion (October 2009), 2092 children were enrolled. Children with HIV (n=45), those whose blood smears lacked species-confirmed P falciparum parasitemia on review by an external, blinded reference parasitology laboratory (n=35), and one child outside of the pre-defined study age range were excluded, leaving 2011 for analysis. Of these, 1078 had UM and 855 had SM, with 48 of the SM (5.6%) being fatal cases. Among those with SM, 850 had either cerebral malaria (CM, n=174), severe malaria anemia (SMA, hemoglobin < 5g/dL, n=558), or lactic acidosis (LA, lactate >5mM, n=482) in an overlapping distribution. The proportion of individuals with Group A or AB increased with categories of increasing disease severity (UM = 28%, SM = 37.1%; fatal = 47.9%). Group O correspondingly decreased (UM = 49.9%, SM = 40.6%, fatal = 37.5%). There was a highly statistically significant difference in the distribution of A, B, AB and O between UM and SM, χ2 =29.57, p=0.000002. Importantly, we observed a significant difference between UM and fatal cases for the distribution of all four ABO groups (χ2 = 4.624, p=0.0315) which was especially significant for Group A or AB versus O (χ2 = 5.946, p=0.0147). A significant difference in ABO distribution was also observed for specific severe malaria syndromes, with Group O enriched for UM and Group A or AB enriched among those with CM (χ2= 12.18, p=0.006791) and SMA (χ2 = 34.24, p Conclusions: This is the largest prospective study on the relationship between ABO blood groups and P falciparum malaria and the first report sufficiently powered to demonstrate the effect of ABO on survival in P falciparum malaria. We report a statistically significant and clinically important advantage among group O hosts and a corresponding disadvantage for group A or AB hosts. Our results confirm clinically the findings of in vitro studies on ABO and cytoadhesion as well as prior retrospective studies on ABO and disease severity. Because our study was conducted in children, the results have direct bearing on the effect of malaria on genetic selection. The results have implications for the pathogenesis of P falciparum malaria, for new treatment strategies aimed at interrupting cytoadhesion, and for our understanding of the worldwide distribution of ABO blood groups. Disclosures: No relevant conflicts of interest to declare.

Background Intercellular adhesion molecule-1 (ICAM-1) is a cytoadhesion molecule implicated in the pathogenesis of Plasmodium falciparum malaria. Elevated levels of soluble ICAM-1 (sICAM-1) have previously been reported with increased malaria disease severity. However, studies have not yet examined both sICAM-1 concentrations and monocyte ICAM-1 expression in the same cohort of patients. To better understand the relationship of soluble and cellular ICAM-1 measurements in malaria, both monocyte ICAM-1 expression and sICAM-1 concentration were measured in children with P. falciparum infection exhibiting a spectrum of clinical severity. Methods Samples were analysed from 160 children, aged 0.5 to 10.8 years, with documented P. falciparum malaria in Kampala, Uganda. The patients belonged to one of three pre-study defined groups: uncomplicated malaria (UM), severe non-fatal malaria (SM-s), and fatal malaria (SM-f). Subset analysis was done on those with cerebral malaria (CM) or severe malaria anaemia (SMA). Monocyte ICAM-1 was measured by flow cytometry. sICAM-1 was measured by enzyme immunoassay. Results Both sICAM-1 and monocyte cell-surface ICAM-1 followed a log-normal distribution. Median sICAM-1 concentrations increased with greater severity-of-illness: 279 ng/mL (UM), 462 ng/mL (SM-s), and 586 ng/mL (SM-f), p < 0.0001. sICAM-1 levels were not statistically different among children with CM compared to SMA. Monocyte ICAM-1 expression was significantly higher in cases of UM compared with SM-s or SM-f (p < 0.001) and was higher among the subset of patients with CM compared with SMA, p < 0.0014. The combination of sICAM-1 and cellular ICAM-1 identified distinct categories of patients (UM with low sICAM-1 and higher monocyte ICAM-1, CM with both sICAM-1 and monocyte ICAM-1 high, and SMA with sICAM-1 high but monocyte ICAM-1 low). Conclusion In this cohort of children with P. falciparum malaria, sICAM-1 levels were associated with severity-of-illness. Patients with UM had higher monocyte ICAM-1 expression consistent with a role for monocyte ICAM-1 in immune clearance during non-severe malaria. Among the subsets of patients with either SMA or CM, monocyte ICAM-1 levels were higher in CM, consistent with the role of ICAM-1 as a marker of cytoadhesion. Categories of disease in pediatric malaria may exhibit specific combinations of soluble and cellular ICAM-1 expression.

Additional file 1: Figure S1. Alterations in the haem axis in children with (A) severe malarial anaemia or (B) cerebral malaria based on disease outcome. There was a trend towards higher plasma levels of haemin and cell-free haemoglobin at presentation in children who died of the infection compared to those who survived; however, these associations are likely underpowered to reach statistical significance (haemin: SMA p = 0.14, CM p = 0.14; haemoglobin: SMA p = 0.06, CM p = 0.25). There was no observable difference between levels of haemopexin and haptoglobin and disease outcome in children with either SMA or CM. The dotted lines indicate the median levels observed in children with UM. Mann–Whitney test.

Full study protocol (including SPIRIT figure): v4.0, dated 06 Feb 2018. (PDF 3510 kb)

Background Malaria is associated with haemolysis and the release of plasma haem. Plasma haem can cause endothelial injury and organ dysfunction, and is normally scavenged by haemopexin to limit toxicity. It was hypothesized that dysregulation of the haem-haemopexin pathway contributes to severe and fatal malaria infections. Methods Plasma levels of haemin (oxidized haem), haemopexin, haptoglobin, and haemoglobin were quantified in a case–control study of Ugandan children with Plasmodium falciparum malaria. Levels at presentation were compared in children with uncomplicated malaria (UM; n = 29), severe malarial anaemia (SMA; n = 27) or cerebral malaria (CM; n = 31), and evaluated for utility in predicting fatal (n = 19) vs non-fatal (n = 39) outcomes in severe disease. A causal role for haemopexin was assessed in a pre-clinical model of experimental cerebral malaria (ECM), following disruption of mouse haemopexin gene (hpx). Analysis was done using Kruskall Wallis tests, Mann–Whitney tests, log-rank tests for survival, and repeated measures ANOVA. Results In Ugandan children presenting with P. falciparum malaria, haemin levels were higher and haemopexin levels were lower in SMA and CM compared to children with UM (haemin, p

Additional file 1: Figure S1. Alterations in the haem axis in children with (A) severe malarial anaemia or (B) cerebral malaria based on disease outcome. There was a trend towards higher plasma levels of haemin and cell-free haemoglobin at presentation in children who died of the infection compared to those who survived; however, these associations are likely underpowered to reach statistical significance (haemin: SMA p = 0.14, CM p = 0.14; haemoglobin: SMA p = 0.06, CM p = 0.25). There was no observable difference between levels of haemopexin and haptoglobin and disease outcome in children with either SMA or CM. The dotted lines indicate the median levels observed in children with UM. Mann–Whitney test.

Introduction To produce health professionals who are oriented towards addressing community priority health needs, the training in medical schools has been transformed to include a component of community-based training. During this period, students spend a part of their training in the communities they are likely to serve upon graduation. They engage and empower local people in the communities to address their health needs during their placements, and at the same time learn from the people. During the community-based component, students are constantly supervised by faculty from the university to ensure that the intended objectives are achieved. The purpose of the present study was to explore student experiences of support supervision from university faculty during their community-based education, research and service (COBERS placements) and to identify ways in which the student learning can be improved through improved faculty supervision. Methods This was a cross-sectional study involving students at the College of Health Sciences, Makerere University, Uganda, who had a community-based component during their training. Data were collected using both questionnaires and focus group discussions. Quantitative data were analyzed using statistical software and thematic approaches were used for the analysis of qualitative data. Results Most students reported satisfaction with the COBERS supervision; however, junior students were less satisfied with the supervision than the more senior students with more experience of community-based training. Although many supervisors assisted students before departure to COBERS sites, a significant number of supervisors made little follow-up while students were in the community. Incorporating the use of information technology avenues such as emails and skype sessions was suggested as a potential way of enhancing supervision amidst resource constraints without faculty physically visiting the sites. Conclusions Although many students were satisfied with COBERS supervision, there are still some challenges, mostly seen with the more junior students. Using information technology could be a solution to some of these challenges.

Full study protocol (including SPIRIT figure): v4.0, dated 06 Feb 2018. (PDF 3123 kb)

Full study protocol (including SPIRIT figure): v4.0, dated 06 Feb 2018. (PDF 3123 kb)