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Few previous studies have reported the effects of glucose-6-phosphate dehydrogenase (G6PD)–deficiency on child health in Africa. We conducted a case-control study in which cases (n = 6829) were children admitted, for any reason, to Kilifi County Hospital, Kenya, while controls (n = 10 179) were recruited from the surrounding community. Cases were subclassified based on their clinical and laboratory findings at admission. We calculated the prevalence of specific diseases by G6PD c.202 genotype, the only significant cause of G6PD-deficiency in this area, then estimated the association between genotype and admission with specific conditions using logistic regression. Among neonates, the prevalence of jaundice was higher in both G6PD c.202T heterozygotes (40/88; 45.5%; P = .004) and homo/hemizygotes (81/134; 60.5%; P < .0001) than in wild-type homozygotes (157/526; 29.9%). Median bilirubin levels also increased across the groups, being highest (239 mmol/L; interquartile range 96-390 mmol/L) in G6PD c.202T homo/hemizygotes. No differences were seen in admission hemoglobin concentrations or the prevalence of anemia or severe anemia by G6PD c.202 genotype. On case control analysis, G6PD heterozygosity was negatively associated with all-cause hospital admission (odds ratio 0.81; 95% confidence interval 0.73-0.90; P < .0001) and, specifically, admission with either pneumonia or Plasmodium falciparum parasitemia; while, conversely, it was positively associated with Gram-positive bacteremia. G6PD c.202T homo/heterozygosity was positively associated with neonatal jaundice, severe pneumonia, the receipt of a transfusion, and in-patient death. Our study supports the conclusion that G6PD c.202T is a balanced polymorphism in which a selective advantage afforded to heterozygous females against malaria is counterbalanced by increased risks of neonatal jaundice, invasive bacterial infections, and anemia.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Severe falciparum malaria has substantially affected human evolution. Genetic association studies of patients with clinically defined severe malaria and matched population controls have helped characterise human genetic susceptibility to severe malaria, but phenotypic imprecision compromises discovered associations. In areas of high malaria transmission, the diagnosis of severe malaria in young children and, in particular, the distinction from bacterial sepsis are imprecise. We developed a probabilistic diagnostic model of severe malaria using platelet and white count data. Under this model, we re-analysed clinical and genetic data from 2220 Kenyan children with clinically defined severe malaria and 3940 population controls, adjusting for phenotype mis-labelling. Our model, validated by the distribution of sickle trait, estimated that approximately one-third of cases did not have severe malaria. We propose a data-tilting approach for case-control studies with phenotype mis-labelling and show that this reduces false discovery rates and improves statistical power in genome-wide association studies., eLife digest In areas of sub-Saharan Africa where malaria is common, most people are frequently exposed to the bites of mosquitoes carrying malaria parasites, so they often have malaria parasites in their blood. Young children, who have not yet built up strong immunity against malaria, often fall ill with severe malaria, a life-threatening disease. It is unclear why some children develop severe malaria and die, while other children with high numbers of parasites in their blood do not develop any apparent symptoms. Genetic susceptibility studies are designed to uncover why such differences exist by comparing individuals with severe malaria (referred to as ‘cases’) with individuals drawn from the general population (known as ‘controls’). But severe malaria can be a challenge to diagnose. Since high numbers of malaria parasites can be found in healthy children, it is sometimes difficult to determine whether the parasites are making a child ill, or whether they are a coincidental finding. Consequently, some of the ‘cases’ recruited into these studies may actually have a different disease, such as bacterial sepsis. This ultimately affects how the studies are interpreted, and introduces error and inaccuracy into the data. Watson, Ndila et al. investigated whether measuring blood biomarkers in patients (derived from the complete blood count, including platelet counts and white blood cell counts) could improve the accuracy with which malaria is diagnosed. They developed a new mathematical model that incorporates platelet and white blood cell counts. This model estimates that in a large cohort of 2,220 Kenyan children diagnosed with severe malaria, around one third of enrolled children did not actually have this disease. Further analysis suggests that patients with severe malaria are highly unlikely to have platelet counts higher than 200,000 per microlitre. This defines a cut-off that researchers can use to avoid recruiting patients who do not have severe malaria in future studies. Additionally, the ability to diagnose severe malaria more accurately can make it easier to detect and treat other diseases with similar symptoms in children with high numbers of malaria parasites in their blood. Watson, Ndila et al.’s findings support the recommendation that all children with suspected malaria be given broad spectrum antibiotics, as many misdiagnosed children will likely have bacterial sepsis. It also suggests that using complete blood counts, which are cheap to obtain and increasingly available in low-resource settings, could improve diagnostic accuracy in future clinical studies of severe malaria. This could ultimately improve the ability of these studies to find new treatments for this life-threatening disease.

Host genetic factors can confer resistance against malaria, raising the question of whether this has led to evolutionary adaptation of parasite populations. In this study we investigated the correlation between host and parasite genetic variation in 4,171 Gambian and Kenya children ascertained with severe malaria due to Plasmodium falciparum. We identified a strong association between sickle haemoglobin (HbS) in the host and variation in three regions of the parasite genome, including nonsynonymous variants in the acyl-CoA synthetase family member PfACS8 on chromosome 2, in a second region of chromosome 2, and in a region containing structural variation on chromosome 11. The HbS-associated parasite alleles are in strong linkage disequilibrium and have frequencies which 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.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Culicine mosquitoes are vectors of human disease-causing pathogens like filarial worms and several arthropod-borne viruses (arboviruses). Currently, there has been an increase in emerging and re-emerging vector-borne diseases along coastal Kenya, which has been of major concern in public health. This study aimed at determining culicine mosquito species abundance, diversity and their host feeding preferences in Taita-Taveta County, Coastal Kenya. Entomological sampling was done during the long-wet season (March and May) and long dry season (June to October) 2016–2018. Mosquito sampling was done using CDC light traps and Backpack aspiration for indoor and outdoor environments. All culicine mosquitoes collected were identified morphologically and categorized according to their physiological status. Blood fed culicine mosquitoes were tested for bloodmeal sources using ELISA. In total, 3,278 culicine mosquitoes were collected, of which 738 (22.5 %) were found indoors and 2,540, (77.5 %) outdoors. The mosquitoes consisted of 18 species belonging to four genera: Aedes (7), Culex (8), Mansonia (2), and Coquillettidia (1). Overall, there was high mosquito species diversity (H) in outdoors (H = 2.4339) than in indoors (H = 2.2523), whereas even distribution (EH) was higher in indoors (EH = 0.9064) than outdoors (EH = 0.8266). Majorly the bloodmeals identified were from multiple host sources with (51.6%), single hosts (41.3%), and unidentified (7.2%). This study has demonstrated a high diversity of culicine mosquitoes with relaxed feeding tendencies. These mosquitoes are contributing to mosquito biting nuisance and the likelihood of exposure of populations to diseases of public health.

Background: Sickle cell disease is the most common severe monogenic disorder in humans. In Africa, 50–90% of children born with sickle cell disease die before they reach their fifth birthday. In this study, we aimed to describe the comparative incidence of specific clinical outcomes among children aged between birth and 5 years with and without sickle cell disease, who were resident within the Kilifi area of Kenya. Methods: This prospective cohort study was done on members of the Kilifi Genetic Birth Cohort Study (KGBCS) on the Indian Ocean coast of Kenya. Recruitment to the study was facilitated through the Kilifi Health and Demographic Surveillance System (KHDSS), which covers a resident population of 260 000 people, and was undertaken between Jan 1, 2006, and April 30, 2011. All children who were born within the KHDSS area and who were aged 3–12 months during the recruitment period were eligible for inclusion. Participants were tested for sickle cell disease and followed up for survival status and disease-specific admission to Kilifi County Hospital by passive surveillance until their fifth birthday. Children with sickle cell disease were offered confirmatory testing and care at a dedicated outpatient clinic. Findings: 15 737 infants were recruited successfully to the KGBCS, and 128 (0·8%) of these infants had sickle cell disease, of whom 70 (54·7%) enrolled at the outpatient clinic within 12 months of recruitment. Mortality was higher in children with sickle cell disease (58 per 1000 person-years of observation, 95% CI 40–86) than in those without sickle cell disease (2·4 per 1000 person-years of observation, 2·0–2·8; adjusted incidence rate ratio [IRR] 23·1, 95% CI 15·1–35·3). Among children with sickle cell disease, mortality was lower in those who enrolled at the clinic (adjusted IRR 0·26, 95% CI 0·11–0·62) and in those with higher levels of haemoglobin F (HbF; adjusted IRR 0·40, 0·17–0·94). The incidence of admission to hospital was also higher in children with sickle cell disease than in children without sickle cell disease (210 per 1000 person-years of observation, 95% CI 174–253, vs 43 per 1000 person-years of observation, 42–45; adjusted IRR 4·80, 95% CI 3·84–6·15). The most common reason for admission to hospital among those with sickle cell disease was severe anaemia (incidence 48 per 1000 person-years of observation, 95% CI 32–71). Admission to hospital was lower in those with a recruitment HbF level above the median (IRR 0·43, 95% CI 0·24–0·78; p=0·005) and those who were homozygous for α-thalassaemia (0·07, 0·01–0·83; p=0·035). Interpretation: Although morbidity and mortality were high in young children with sickle cell disease in this Kenyan cohort, both were reduced by early diagnosis and supportive care. The emphasis must now move towards early detection and prevention of long-term complications of sickle cell disease.

Background Many parts of Africa have witnessed reductions in Plasmodium falciparum transmission over the last 15 years. Since immunity to malaria is acquired more rapidly at higher transmission, the slower acquisition of immunity at lower transmission may partially offset the benefits of reductions in transmission. We examined the clinical spectrum of disease and predictors of mortality after sustained changes in transmission intensity, using data collected from 1989 to 2016. Methods We conducted a temporal observational analysis of 18,000 children, aged 14 days to 14 years old, who were admitted to Kilifi County Hospital, Kenya, from 1989 to 2016 with malaria. We describe the trends over time of the clinical and laboratory criteria for severe malaria and associated risk of mortality. Results During the time periods 1989–2003, 2004–2008, and 2009–2016, Kilifi County Hospital admitted averages of 657, 310, and 174 cases of severe malaria per year including averages of 48, 14, and 12 malaria-associated deaths per year, respectively. The median ages in years of children admitted with cerebral malaria, severe anaemia, and malaria-associated mortality were 3.0 (95% confidence interval (CI) 2.2–3.9), 1.1 (95% CI 0.9–1.4), and 1.1 (95% CI 0.3–2.2) in the year 1989, rising to 4.9 (95% CI 3.9–5.9), 3.8 (95% CI 2.5–7.1), and 5 (95% CI 3.3–6.3) in the year 2016. The ratio of children with cerebral malaria to severe anaemia rose from 1:2 before 2004 to 3:2 after 2009. Hyperparasitaemia was a risk factor for death after 2009 but not in earlier time periods. Conclusion Despite the evidence of slower acquisition of immunity, continued reductions in the numbers of cases of severe malaria resulted in lower overall mortality. Our temporal data are limited to a single site, albeit potentially applicable to a secular trend present in many parts of Africa. Electronic supplementary material The online version of this article (10.1186/s12916-019-1359-9) contains supplementary material, which is available to authorized users.

Sickle cell anemia (SCA) is the commonest severe monogenic disorders of humans. The disease has been highly characterized in high‐income countries but not in sub‐Saharan Africa where SCA is most prevalent. We conducted a retrospective cohort study of all children 0–13 years admitted from within a defined study area to Kilifi County Hospital in Kenya over a five‐year period. Children were genotyped for SCA retrospectively and incidence rates calculated with reference to population data. Overall, 576 of 18,873 (3.1%) admissions had SCA of whom the majority (399; 69.3%) were previously undiagnosed. The incidence of all‐cause hospital admission was 57.2/100 person years of observation (PYO; 95%CI 52.6–62.1) in children with SCA and 3.7/100 PYO (95%CI 3.7–3.8) in those without SCA (IRR 15.3; 95%CI 14.1–16.6). Rates were higher for the majority of syndromic diagnoses at all ages beyond the neonatal period, being especially high for severe anemia (hemoglobin

Supplemental Digital Content is Available in the Text., Introduction: HIV-1 is transmitted through semen from men to their sexual partners. Genital infections can increase HIV-1 RNA shedding in semen, but shedding also occurs in the absence of typical pathogens. We hypothesized that higher bacterial concentrations in semen would be associated with higher HIV-1 RNA levels. Methods: We analyzed semen samples from 42 HIV-1–seropositive Kenyan men using quantitative polymerase chain reaction (PCR) to assess bacterial concentrations and real-time PCR to measure HIV-1 RNA levels. Generalized estimation equations were used to evaluate associations between these 2 measures. Broad-range 16S rRNA gene PCR with pyrosequencing was performed on a subset of 13 samples to assess bacterial community composition. Results: Bacteria were detected in 96.6% of 88 samples by quantitative PCR. Semen bacterial concentration and HIV-1 RNA levels were correlated 0.30 (P = 0.01). The association between bacterial concentration and HIV-1 RNA detection was not significant after adjustment for antiretroviral therapy (ART) (adjusted odds ratio: 1.27, 95% CI: 0.84 to 1.91). Factors associated with semen bacterial concentration included insertive anal sex (adjusted beta 0.92, 95% CI: 0.12 to 1.73) and ART use (adjusted beta: −0.77, 95% CI: −1.50 to 0.04). Among 13 samples with pyrosequencing data, Corynebacterium spp., Staphylococcus spp., and Streptococcus spp. were most frequently detected. Conclusion: Most of these HIV-1–infected men had bacteria in their semen. ART use was associated with undetectable semen HIV-1 RNA and lower semen bacterial concentrations, whereas insertive anal sex was associated with higher bacterial concentrations. Additional studies evaluating the relationship between semen bacteria, inflammation, mucosal immunity, and HIV-1 shedding are needed to understand implications for HIV-1 transmission.

Many human genetic associations with resistance to malaria have been reported, but few have been reliably replicated. We collected data on 11,890 cases of severe malaria due to Plasmodium falciparum and 17,441 controls from 12 locations in Africa, Asia and Oceania. We tested 55 SNPs in 27 loci previously reported to associate with severe malaria. There was evidence of association at P < 1 × 10(-4) with the HBB, ABO, ATP2B4, G6PD and CD40LG loci, but previously reported associations at 22 other loci did not replicate in the multicenter analysis. The large sample size made it possible to identify authentic genetic effects that are heterogeneous across populations or phenotypes, with a striking example being the main African form of G6PD deficiency, which reduced the risk of cerebral malaria but increased the risk of severe malarial anemia. The finding that G6PD deficiency has opposing effects on different fatal complications of P. falciparum infection indicates that the evolutionary origins of this common human genetic disorder are more complex than previously supposed.

INTRODUCTION Malaria parasites cause human disease by invading and replicating inside red blood cells. In the case of Plasmodium falciparum , this can lead to severe forms of malaria that are a major cause of childhood mortality in Africa. This species of parasite enters the red blood cell through interactions with surface proteins including the glycophorins GYPA and GYPB, which determine the polymorphic MNS blood group system. In a recent genome-wide association study, we identified alleles associated with protection against severe malaria near the cluster of genes encoding these invasion receptors. RATIONALE Investigation of genetic variants at this locus and their relation to severe malaria is challenging because of the high sequence similarity between the neighboring glycophorin genes and the relative lack of available sequence data capturing the genetic diversity of sub-Saharan Africa. To better assess whether variation in the glycophorin genes could explain the signal of association, we generated additional sequence data from sub-Saharan African populations and developed an analytical approach to characterize structural variation at this complex locus. RESULTS Using 765 newly sequenced human genomes from 10 African ethnic groups along with data from the 1000 Genomes Project, we generated a reference panel of haplotypes across the glycophorin region. In addition to single-nucleotide polymorphisms and short indels, we assayed large copy number variants (CNVs) using sequencing read depth and uncovered extensive structural diversity. By imputing from this reference panel into 4579 severe malaria cases and 5310 controls from three African populations, we found that a complex CNV, here called DUP4, is associated with resistance to severe malaria and fully explains the previously reported signal of association. In our sample, DUP4 is present only in east Africa, and this localization, as well as the extent of similarity between DUP4 haplotypes, suggests that it has recently increased in frequency, presumably under natural selection due to malaria. To evaluate the potential functional consequences of this structural variant, we analyzed high-coverage sequence-read data from multiple individuals to generate a model of the DUP4 chromosome structure. The DUP4 haplotype contains five glycophorin genes, including two hybrid genes that juxtapose the extracellular domain of GYPB with the transmembrane and intracellular domains of GYPA. Noting that these predicted hybrids are characteristic of the Dantu antigen in the MNS blood group system, we sequenced a Dantu positive individual and confirmed that DUP4 is the molecular basis of the Dantu NE blood group variant. CONCLUSION Although a role for GYPA and GYPB in parasite invasion is well known, a direct link between glycophorin polymorphisms and clinical susceptibility to malaria has been elusive. Here we have provided a systematic catalog of CNVs, describing structural diversity that may have functional importance at this locus. Our results identify a specific variant that encodes hybrid glycophorin proteins and is associated with protection against severe malaria. This discovery calls for further work to determine how this particular molecular rearrangement affects parasite invasion and the red blood cell response and may lead us toward new parasite vulnerabilities that can be utilized in future interventions against this deadly disease.

BackgroundEACCR2 is an EDCTP-funded, Eastern African-led network established in May 2009, with 23 regional partners from Ehtiopia, Kenya, Sudan, Tanzania, and Uganda, and 8 northern partners from Germany, Netherlands, Norway, Sweden and United Kingdom. The objective is to strengthen capacity to conduct health research to international standards with specific focus on clinical trials on poverty-relevant diseases such as HIV, TB, malaria and neglected infectious diseases. EACCR2 optimises the use of shared research infrastructures and other regional capacity building resources and opportunities.ActivitiesThe activities of the network are implemented in five work packages cutting across ‘nodes’ in different countries. Capacity building programmes and studies funded by EDCTP are implemented by coordinators at the disease nodes. The Uganda Virus Research Institute (UVRI) hosts the secretariat of the consortium of five nodes located in the following institutions: Malaria Node in Kilifi-Kenya Medical Research Institute Wellcome Trust, Tanzania; Training Node in Kilimanjaro Clinical Research Institute, Tanzania; Tuberculosis Node at the National Institute of Medical Research- Muhimbili, Tanzania; the Neglected and Re-Emerging Tropical Diseases Node at the University of Khartoum, Sudan; and the HIV Node at UVRI, Uganda.Coordinators form the project implementation committee which meets via skype or teleconference every quarter to assess progress, share best practice and challenges of the network. Scientific and annual meetings are arranged every year in one of the implementing institutions. During such meetings, students, the nodes and steering committee also meet to minimise travel costs while helping teams to network.EACCR2 learns from the experiences, best practice and challenges of EACCR1 while implementing its current activities. Careful planning and consensus building from all partners has been the driving force to build and implement activities of this virtual network. EACCR2 also works closely with other EDCTP Networks of Excellence, i.e. in Central Africa (CANTAM), West Africa (WANETAM), and Southern Africa (TESA).

The mechanisms of death and neurologic sequelae in African children with cerebral malaria are undetermined. Because pathologic features are confined to the cerebral vasculature, perturbations in cerebral hemodynamics may be responsible. We compared the transcranial Doppler findings in 50 children with cerebral malaria with those of 115 conscious Kenyan children. In addition, 10 children with cerebral malaria were studied during intracranial pressure monitoring and nine children were studied during the agonal stages. In the children with cerebral malaria, cerebral blood flow velocity was increased in 30%, usually associated with seizures. Of the 11 children who developed neurologic sequelae, six had sonographic abnormalities associated with lateralizing deficits, including four children with hemiparesis (in two children the contralateral middle cerebral artery could not be insonated and two had transient increases in blood flow velocity associated with seizures). In the children with severe intracranial hypertension, there was a significant linear relationship between the cerebral perfusion pressure and blood flow velocity, suggesting that autoregulation was impaired. Sonographic features of progressive intracranial hypertension, were observed in three children with cerebral malaria who died. Perturbations of cerebral hemodynamics are associated with a poor outcome in Kenyan children with cerebral malaria.

Background With strict adherence to international recommended treatment guidelines, the case fatality for severe malnutrition ought to be less than 5%. In African hospitals, fatality rates of 20% are common and are often attributed to poor training and faulty case management. Improving outcome will depend upon the identification of those at greatest risk and targeting limited health resources. We retrospectively examined the major risk factors associated with early (, A retrospective examination of major risk factors associated with in-hospital deaths in children with severe malnutrition has identified admission features that could help distinguish those at highest risk., Editors' Summary Background. Severe malnutrition is thought to be responsible, at least in part, for a large proportion of the many millions of deaths every year among children below the age of five years. The World Health Organization (WHO) has developed guidelines for management of the severely malnourished child in the hospital. These guidelines outline ten initial steps for routine care, followed by treatment of associated conditions and rehabilitation. However, death rates among children admitted to hospital with severe malnutrition are worryingly high, commonly 20% or sometimes even higher. Many hospitals have reported that following introduction of the WHO guidelines, the death rates have been cut, but not to a level that the WHO defines as acceptable (5% or lower). Why Was This Study Done? In the region where this study was done, an area on the coast of Kenya, East Africa, malnutrition is very common. The local hospital, Kilifi District Hospital, currently reports a death rate of approximately 19% among children admitted with severe malnutrition, even with implementation of the WHO guidelines. A group of researchers based at the hospital wanted to see if they could identify those children who were most likely to die. Their aim was to see which aspects of the children's medical condition put them at highest risk. This information would be useful in ensuring that high-risk children received the most appropriate care. What Did the Researchers Do and Find? The researchers studied all severely malnourished children over three months of age who were admitted to the Kilifi District Hospital between September 2000 and June 2002. The children were treated according to the WHO guidelines, and the research group collected data on the condition of the children after treatment (their “outcomes”), as well as for relevant clinical signs and symptoms. The study involved 920 children, of whom 176 died in hospital (a death rate of 19%). They then examined the data to see which characteristics on admission were associated with early death (less than 48 h) and later deaths. They found that four clinical features, which could be easily ascertained at the bedside on admission, were associated with a large proportion of the early deaths. These four signs were slow heart rate, weak pulse volume, depressed consciousness level, and a delayed capillary refilling time (as tested by pressing a fingernail bed to blanche the finger, releasing it, and observing the time taken to reperfuse the capillaries—or recolor the nailbed). The researchers proposed that these findings, together with a number of other features that were associated with the later deaths could be used to identify three groups of patients differing in their need for emergency care: a high-risk group (with any of the four signs listed above, or hypoglycemia, and among whom mortality was 34%); a moderate-risk group (among whom mortality was 23%); and a low-risk group (mortality 7%). What Do These Findings Mean? First, the death rate amongst these children was very high even though WHO guidelines were used to guide management. The signs reported here as indicators of poor outcome may prove useful in future in identifying high-risk individuals to ensure they receive the right treatment. However, the indicators proposed here would need further evaluation before current guidelines for treatment of the severely malnourished child could be changed. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030500. • Information on severe malnutrition is available from the World Health Organization • Management guidelines from the WHO can also be downloaded in many languages • UNICEF, the United Nations Children's Fund, provides relevant resources and statistics as well as information about its programs addressing malnutrition worldwide • Information from Médecins Sans Frontières (MSF) on acute malnutrition worldwide and MSF's response to current emergencies

Small hospitals sit at the apex of the pyramid of primary care in the health systems of many low-income countries. If the Millennium Development Goal for child survival is to be achieved, hospital care for referred severely ill children will need to be improved considerably in parallel with primary care in many countries. Yet little is known about how to achieve this. This article describes the evolution and final design of an intervention study that is attempting to improve hospital care for children in Kenyan district hospitals. It illustrates many of the difficulties involved in reconciling epidemiological rigour and feasibility in studies at a health system, rather than an individual, level and the importance of the depth and breadth of analysis when trying to provide a plausible answer to the question: does it work? Although there are increasing calls for more health systems research in low-income countries, the importance of strong, broadly based local partnerships and long-term commitment even to initiate projects is not always appreciated.

Objectives: Genitourinary tract samples are required to investigate male HIV-1 infectivity. Because semen collection is often impractical, the acceptability, feasibility and validity of post-prostatic massage fluid/ urine (post-PMF/U) was evaluated for studying male genitourinary HIV-1 shedding. Methods: HIV-1-seropositive men were evaluated after 48 h of sexual abstinence. At each visit, a clinician performed prostatic massage, then post-PMF/U and blood were collected. Participants provided semen specimens 1 week later. An audio computer-assisted self-interview (ACASI) administered after each specimen collection evaluated acceptability, adherence to instructions and recent genitourinary symptoms. HIV-1 RNA was quantified using a real-time PCR assay. Detection and quantitation of HIV-1 RNA and stability over visits were compared for semen, post-PMF/U and blood. Results: Post-PMF/U was successfully obtained at 106 visits (64%) and semen at 136 visits (81%, p

Background The emergence of artemisinin-resistant P. falciparum malaria in South-East Asia highlights the need for continued global surveillance of the efficacy of artemisinin-based combination therapies. Methods On the Kenyan coast we studied the treatment responses in 474 children 6–59 months old with uncomplicated P. falciparum malaria in a randomized controlled trial of dihydroartemisinin-piperaquine vs. artemether-lumefantrine from 2005 to 2008. (ISRCTN88705995) Results The proportion of patients with residual parasitemia on day 1 rose from 55% in 2005–2006 to 87% in 2007–2008 (odds ratio, 5.4, 95%CI, 2.7–11.1; P37.5°C, 2.8, 1.9–4.1; P

BACKGROUND: The candidate malaria vaccine RTS,S/AS01E has entered phase 3 trials, but data on long-term outcomes are limited. METHODS: For 4 years, we followed children who had been randomly assigned, at 5 to 17 months of age, to receive three doses of RTS,S/AS01E vaccine (223 children) or rabies vaccine (224 controls). The end point was clinical malaria (temperature of ≥37.5°C and Plasmodium falciparum parasitemia density of >2500 parasites per cubic millimeter). Each child's exposure to malaria was estimated with the use of the distance-weighted local prevalence of malaria. RESULTS: Over a period of 4 years, 118 of 223 children who received the RTS,S/AS01E vaccine and 138 of 224 of the controls had at least 1 episode of clinical malaria. Vaccine efficacies in the intention-to-treat and per-protocol analyses were 29.9% (95% confidence interval [CI], 10.3 to 45.3; P=0.005) and 32.1% (95% CI, 11.6 to 47.8; P=0.004), respectively, calculated by Cox regression. Multiple episodes were common, with 551 and 618 malarial episodes in the RTS,S/AS01E and control groups, respectively; vaccine efficacies in the intention-to-treat and per-protocol analyses were 16.8% (95% CI, -8.6 to 36.3; P=0.18) and 24.3% (95% CI, 1.9 to 41.6; P=0.04), respectively, calculated by the Andersen-Gill extension of the Cox model. For every 100 vaccinated children, 65 cases of clinical malaria were averted. Vaccine efficacy declined over time (P=0.004) and with increasing exposure to malaria (P=0.001) in the per-protocol analysis. Vaccine efficacy was 43.6% (95% CI, 15.5 to 62.3) in the first year but was -0.4% (95% CI, -32.1 to 45.3) in the fourth year. Among children with a malaria-exposure index that was average or lower than average, the vaccine efficacy was 45.1% (95% CI, 11.3 to 66.0), but among children with a malaria-exposure index that was higher than average it was 15.9% (95% CI, -11.0 to 36.4). CONCLUSIONS: The efficacy of RTS,S/AS01E vaccine over the 4-year period was 16.8%. Efficacy declined over time and with increasing malaria exposure. (Funded by the PATH Malaria Vaccine Initiative and Wellcome Trust; ClinicalTrials.gov number, NCT00872963.).

Background. Human immunodeficiency virus (HIV) infection, malnutrition, and invasive bacterial infection (IBI) are reported among children with severe malaria. However, it is unclear whether their cooccurrence with falciparum parasitization and severe disease happens by chance or by association among children in areas where malaria is endemic. Methods. We examined 3068 consecutive children admitted to a Kenyan district hospital with clinical features of severe malaria and 592 control subjects from the community. We performed multivariable regression analysis, with each case weighted for its probability of being due to falciparum malaria, using estimates of the fraction of severe disease attributable to malaria at different parasite densities derived from cross-sectional parasitological surveys of healthy children from the same community. Results. HIV infection was present in 133 (12%) of 1071 consecutive parasitemic admitted children (95% confidence interval [CI], 11%–15%). Parasite densities were higher in HIV-infected children. The odds ratio for admission associated with HIV infection for admission with true severe falciparum malaria was 9.6 (95% CI, 4.9– 19); however, this effect was restricted to children aged ≥1 year. Malnutrition was present in 507 (25%) of 2048 consecutive parasitemic admitted children (95% CI, 23%–27%). The odd ratio associated with malnutrition for admission with true severe falciparum malaria was 4.0 (95% CI, 2.9–5.5). IBI was detected in 127 (6%) of 2048 consecutive parasitemic admitted children (95% CI, 5.2%–7.3%). All 3 comorbidities were associated with increased case fatality. Conclusions. HIV, malnutrition and IBI are biologically associated with severe disease due to falciparum malaria rather than being simply alternative diagnoses in co-incidentally parasitized children in an endemic area.

Objective: The objective was to measure the efficacy of the vaccination regimen FFM ME-TRAP in preventing episodes of clinical malaria among children in a malaria endemic area. FFM ME-TRAP is sequential immunisation with two attenuated poxvirus vectors (FP9 and modified vaccinia virus Ankara), which both deliver the pre-erythrocytic malaria antigen construct multiple epitope–thrombospondin-related adhesion protein (ME-TRAP). Design: The trial was randomised and double-blinded. Setting: The setting was a rural, malaria-endemic area of coastal Kenya. Participants: We vaccinated 405 healthy 1- to 6-year-old children. Interventions: Participants were randomised to vaccination with either FFM ME-TRAP or control (rabies vaccine). Outcome Measures: Following antimalarial drug treatment children were seen weekly and whenever they were unwell during nine months of monitoring. The axillary temperature was measured, and blood films taken when febrile. The primary analysis was time to a parasitaemia of over 2,500 parasites/μl. Results: The regime was moderately immunogenic, but the magnitude of T cell responses was lower than in previous studies. In intention to treat (ITT) analysis, time to first episode was shorter in the FFM ME-TRAP group. The cumulative incidence of febrile malaria was 52/190 (27%) for FFM ME-TRAP and 40/197 (20%) among controls (hazard ratio = 1.52). This was not statistically significant (95% confidence interval [CI] 1.0–2.3; p = 0.14 by log-rank). A group of 346 children were vaccinated according to protocol (ATP). Among these children, the hazard ratio was 1.3 (95% CI 0.8–2.1; p = 0.55 by log-rank). When multiple malaria episodes were included in the analyses, the incidence rate ratios were 1.6 (95% CI 1.1–2.3); p = 0.017 for ITT, and 1.4 (95% CI 0.9–2.1); p = 0.16 for ATP. Haemoglobin and parasitaemia in cross-sectional surveys at 3 and 9 mo did not differ by treatment group. Among children vaccinated with FFM ME-TRAP, there was no correlation between immunogenicity and malaria incidence. Conclusions: No protection was induced against febrile malaria by this vaccine regimen. Future field studies will require vaccinations with stronger immunogenicity in children living in malarious areas., Editorial Commentary Background: Malaria kills over a million people a year worldwide, and young children in sub-Saharan are particularly at risk. Cheap, safe, and effective vaccines are needed. One strategy involves a double-vaccination process. This approach (termed “prime-boost”) uses two different delivery methods to transmit the same antigen (part of a protein from the malaria parasite that can trigger an immune response). The two-step vaccination is designed to achieve a greater immune response than with just one vaccination. One research group, based in Oxford in the UK, is using an antigen called “ME-TRAP,” which is delivered using first a strain of modified fowlpox virus (called FP9), then a weakened vaccinia virus (called MVA). Previous studies done in adult UK volunteers have been promising, achieving an immune response and some protection against malaria when volunteers were deliberately infected. However, this approach has not been tested in the group most in need of a vaccine—young African children. Therefore a field trial was conducted among 405 healthy children aged 1–6 years, in a region of Kenya with year-round malaria transmission. Children were randomized to receive either the sequence of vaccines delivering ME-TRAP or to receive a rabies vaccine (as control, but which still gives the children some benefit for taking part in the trial). The children were followed up for nine months, and the primary aim of the trial was to compare the occurrence of clinical malaria (fever combined with malaria parasites in the blood) in the two groups. What this trial shows: In the 387 children receiving vaccine and having at least one follow-up visit the vaccine did produce an immune response; however, this immune response did not seem to be protective, as the occurrence of malaria was slightly higher in the group receiving the candidate vaccine—although this difference was not statistically significant. Safety data were also collected; the number and severity of adverse events were similar between volunteers receiving the rabies vaccine and those receiving the candidate malaria vaccine, and any serious events were not judged to be linked to the vaccines by the trial's data safety monitoring board. Strengths and limitations: The methods used in the trial were robust, using appropriate randomization procedures and blinding of participants and researchers. Outcome measures (clinical malaria, defined as fever together with parasites in the blood over 2,500/microliter) were clinically relevant. In order to detect cases of malaria in vaccinated children, health workers visited children weekly, and children with a temperature over 37.5 °C were tested for parasites in the blood. (In between the weekly visits, self-report and referral for assessment also allowed detection of cases.) This process of active detection of malaria cases (as opposed to obtaining data on clinical malaria only from self-report or referral) enables a smaller sample size to be used in the trial, but it is not clear whether this approach is more or less specific at picking up malaria cases than are passive methods. The researchers aimed to ensure that their case detection methods were specific; for children with normal temperature, but reported by their parents as feverish, parasite tests were done only if subsequent temperature readings were high. Contribution to the evidence: Previous studies of ME-TRAP using the FP9 and MVA vectors have shown the candidate vaccine is safe and induces a strong immune response. The safety result was also supported by the findings of the current trial, conducted in young Kenyan children, but a 5-fold lower immune response was found compared to previous studies. The trial showed that this weak immune response was not effective at preventing clinical cases of malaria in this group of children, although it is not clear why the immune response was lower than expected.

Over the last decade, a number of observations have suggested that platelets may play a role in the pathophysiology of severe malaria. However, somewhat paradoxically, thrombocytopenia is not associated clearly with outcome. We studied the relationship between thrombocytopenia and cytokines in Kenyan children with severe malaria and showed that thrombocytopenia (platelet count < 150 x 10(9)/L) strongly correlates with high levels of interleukin (IL)-10. Several studies have shown that high levels of IL-10 are associated with a favorable outcome in severe malaria. Taken together, these data suggest why thrombocytopenia has a complex relationship with severe disease and suggest one mechanism whereby IL-10 may modify the outcome of severe disease.

A heterologous prime-boost strategy has been developed to potently induce T cell responses to pre-erythrocytic malaria antigens. Efficacy in the field is likely to depend on both peak immunogenicity and the durability of responses. To improve both immunogenicity and durability of responses, 54 adult males from a malaria endemic area were immunized with different vaccination regimens, systematically varying antigenic insert and the number and sequence of component vaccinations. The component vaccinations were recombinant attenuated viruses, either fowlpox (FP) 9 or modified vaccinia virus Ankara (MVA). These were recombinant for either of two pre-erythrocytic malaria antigens (multiple epitope-thrombospondin-related adhesion protein, ME-TRAP, or circumsporozoite antigen (CS). ELISPOT assays were used to measure the effector and resting memory T cell responses. Sequence, antigen insert and number of vaccinations influenced immunogenicity, but the novel alternating vector immunizations generated the largest resting memory T cell populations. Effector responses were maintained at 84% of the peak response after 270 days. This durability of response is unprecedented. Classical prime-boost vaccination responses were at 5% of the peak after 270 days. Vaccines administered by heterologous prime-boost regimes are being developed for diverse pathogens and cancer. These data suggest these vaccines should also be administered by alternating vector regimens in clinical development.