Clearance of Asymptomatic P. falciparum Infections Interacts with the Number of Clones to Predict the Risk of Subsequent Malaria in Kenyan Children

BACKGROUND: Protective immunity to malaria is acquired after repeated infections in endemic areas. Asymptomatic multiclonal P. falciparum infections are common and may predict host protection. Here, we have investigated the effect of clearing asymptomatic infections on the risk of clinical malaria. METHODS: Malaria episodes were continuously monitored in 405 children (1-6 years) in an area of moderate transmission, coastal Kenya. Blood samples collected on four occasions were assessed by genotyping the polymorphic P. falciparum merozoite surface protein 2 using fluorescent PCR and capillary electrophoresis. Following the second survey, asymptomatic infections were cleared with a full course of dihydroartemisinin. RESULTS: Children who were parasite negative by PCR had a lower risk of subsequent malaria regardless of whether treatment had been given. Children with ≥ 2 clones had a reduced risk of febrile malaria compared with 1 clone after clearance of asymptomatic infections, but not if asymptomatic infections were not cleared. Multiclonal infection was associated with an increased risk of re-infection after drug treatment. However, among the children who were re-infected, multiclonal infections were associated with a shift from clinical malaria to asymptomatic parasitaemia. CONCLUSION: The number of clones was associated with exposure as well as blood stage immunity. These effects were distinguished by clearing asymptomatic infection with anti-malarials. Exposure to multiple P. falciparum infections is associated with protective immunity, but there appears to be an additional effect in untreated multiclonal infections that offsets this protective effect

Helminth Infection and Eosinophilia and the Risk of Plasmodium falciparum Malaria in 1- to 6-Year-Old Children in a Malaria Endemic Area

Malaria infection and other parasitic infections are widespread in developing countries. There is evidence from some studies that intestinal worm infections may increase the risk of developing febrile malaria. However, the evidence is mixed, and some studies have found no effect or even protective effects. A vaccine trial was recently conducted to assess the efficacy of a candidate malaria vaccine. Episodes of malaria were monitored. The vaccine was not protective, but data was also recorded on the prevalence of worm infections. The rates of febrile malaria did not seem to vary according to worm infection in this study. However, because of the relatively low prevalence of worm infection, the study did not have high power. Given the conflicting findings in the literature, and the potential for the effect of worm infection to vary geographically, it is important that larger, definitive studies are conducted, since even quite small effects might be important for global public health

Lactate levels in severe malarial anaemia are associated with haemozoin-containing neutrophils and low levels of IL-12

BACKGROUND: Hyperlactataemia is often associated with a poor outcome in severe malaria in African children. To unravel the complex pathophysiology of this condition the relationship between plasma lactate levels, parasite density, pro- and anti-inflammatory cytokines, and haemozoin-containing leucocytes was studied in children with severe falciparum malarial anaemia. METHODS: Twenty-six children with a primary diagnosis of severe malarial anaemia with any asexual Plasmodium falciparum parasite density and Hb < 5 g/dL were studied and the association of plasma lactate levels and haemozoin-containing leucocytes, parasite density, pro- and anti-inflammatory cytokines was measured. The same associations were measured in non-severe malaria controls (N = 60). RESULTS: Parasite density was associated with lactate levels on admission (r = 0.56, P < 0.005). Moreover, haemozoin-containing neutrophils and IL-12 were strongly associated with plasma lactate levels, independently of parasite density (r = 0.60, P = 0.003 and r = -0.46, P = 0.02, respectively). These associations were not found in controls with uncomplicated malarial anaemia. CONCLUSION: These data suggest that blood stage parasites, haemozoin and low levels of IL-12 may be associated with the development of hyperlactataemia in severe malarial anaemia

Defining childhood severe falciparum malaria for intervention studies.

Background Clinical trials of interventions designed to prevent severe falciparum malaria in children require a clear endpoint. The internationally accepted definition of severe malaria is sensitive, and appropriate for clinical purposes. However, this definition includes individuals with severe nonmalarial disease and coincident parasitaemia, so may lack specificity in vaccine trials. Although there is no “gold standard” individual test for severe malaria, malaria-attributable fractions (MAFs) can be estimated among groups of children using a logistic model, which we use to test the suitability of various case definitions as trial endpoints. Methods and Findings A total of 4,583 blood samples were taken from well children in cross-sectional surveys and from 1,361 children admitted to a Kenyan District hospital with severe disease. Among children under 2 y old with severe disease and over 2,500 parasites per microliter of blood, the MAFs were above 85% in moderate- and low-transmission areas, but only 61% in a high-transmission area. HIV and malnutrition were not associated with reduced MAFs, but gastroenteritis with severe dehydration (defined by reduced skin turgor), lower respiratory tract infection (clinician's final diagnosis), meningitis (on cerebrospinal fluid [CSF] examination), and bacteraemia were associated with reduced MAFs. The overall MAF was 85% (95% confidence interval [CI] 83.8%–86.1%) without excluding these conditions, 89% (95% CI 88.4%–90.2%) after exclusions, and 95% (95% CI 94.0%–95.5%) when a threshold of 2,500 parasites/μl was also applied. Applying a threshold and exclusion criteria reduced sensitivity to 80% (95% CI 77%–83%). Conclusions The specificity of a case definition for severe malaria is improved by applying a parasite density threshold and by excluding children with meningitis, lower respiratory tract infection (clinician's diagnosis), bacteraemia, and gastroenteritis with severe dehydration, but not by excluding children with HIV or malnutrition

Homozygosity and risk of childhood death due to invasive bacterial disease

BACKGROUND: Genetic heterozygosity is increasingly being shown to be a key predictor of fitness in natural populations, both through inbreeding depression, inbred individuals having low heterozygosity, and also through chance linkage between a marker and a gene under balancing selection. One important component of fitness that is often highlighted is resistance to parasites and other pathogens. However, the significance of equivalent loci in human populations remains unclear. Consequently, we performed a case-control study of fatal invasive bacterial disease in Kenyan children using a genome-wide screen with microsatellite markers. METHODS: 148 cases, comprising children aged <13 years who died of invasive bacterial disease, (variously, bacteraemia, bacterial meningitis or neonatal sepsis) and 137 age-matched, healthy children were sampled in a prospective study conducted at Kilifi District Hospital, Kenya. Samples were genotyped for 134 microsatellite markers using the ABI LD20 marker set and analysed for an association between homozygosity and mortality. RESULTS: At five markers homozygosity was strongly associated with mortality (odds ratio range 4.7 - 12.2) with evidence of interactions between some markers. Mortality was associated with different non-overlapping marker groups in Gram positive and Gram negative bacterial disease. Homozygosity at susceptibility markers was common (prevalence 19-49%) and, with the large effect sizes, this suggests that bacterial disease mortality may be strongly genetically determined. CONCLUSION: Balanced polymorphisms appear to be more widespread in humans than previously appreciated and play a critical role in modulating susceptibility to infectious disease. The effect sizes we report, coupled with the stochasticity of exposure to pathogens suggests that infection and mortality are far from random due to a strong genetic basis

Homozygosity and risk of childhood death due to invasive bacterial disease.

BACKGROUND: Genetic heterozygosity is increasingly being shown to be a key predictor of fitness in natural populations, both through inbreeding depression, inbred individuals having low heterozygosity, and also through chance linkage between a marker and a gene under balancing selection. One important component of fitness that is often highlighted is resistance to parasites and other pathogens. However, the significance of equivalent loci in human populations remains unclear. Consequently, we performed a case-control study of fatal invasive bacterial disease in Kenyan children using a genome-wide screen with microsatellite markers. METHODS: 148 cases, comprising children aged <13 years who died of invasive bacterial disease, (variously, bacteraemia, bacterial meningitis or neonatal sepsis) and 137 age-matched, healthy children were sampled in a prospective study conducted at Kilifi District Hospital, Kenya. Samples were genotyped for 134 microsatellite markers using the ABI LD20 marker set and analysed for an association between homozygosity and mortality. RESULTS: At five markers homozygosity was strongly associated with mortality (odds ratio range 4.7 - 12.2) with evidence of interactions between some markers. Mortality was associated with different non-overlapping marker groups in Gram positive and Gram negative bacterial disease. Homozygosity at susceptibility markers was common (prevalence 19-49%) and, with the large effect sizes, this suggests that bacterial disease mortality may be strongly genetically determined. CONCLUSION: Balanced polymorphisms appear to be more widespread in humans than previously appreciated and play a critical role in modulating susceptibility to infectious disease. The effect sizes we report, coupled with the stochasticity of exposure to pathogens suggests that infection and mortality are far from random due to a strong genetic basis

Malaria vector bionomics in Taita-Taveta County, coastal Kenya

Background Estimation of the composition and densities of mosquito species populations is crucial for monitoring the epidemiology of mosquito-borne diseases and provide information on local vectors to public health officials and policy-makers. The aim of this study was to evaluate malaria vector bionomics in ecologically distinct sites in Taita-Taveta County, Kenya. Methods Adult mosquitoes were collected using backpack aspirators and paired indoor/outdoor CDC light traps in 10 randomly selected households in six villages with distinct ecologies over a study period of 3 years. All Anopheles mosquitoes were morphotyped, and sibling species of Anopheles gambiae sensu lato (An. gambiae s.l.) were identified and separated by PCR analysis of extracted ribosomal DNA. All female anophelines were tested for sporozoite infectivity, with engorged females screened for blood-meal sources using the enzyme-linked immunosorbent assay technique. A subsample of those testing positive and those testing negative for Plasmodium in the ELISA were subjected to PCR assay. Results A total of eight different Anopheles species were collected both indoors and outdoors. Anopheles gambiae s.l. (82.6%, n = 5252) was the predominant species sensu lato, followed by Anopheles coustani sensu lato (An. coustani s.l.; (10.5%, n = 666) and Anopheles funestus sensu lato (An. funestus s.l.; 5.6%, n = 357). A subset of 683 mosquito samples representing An. gambiae s.l. (n = 580, approx. 11.0%) and An. funestus s.l. (n = 103, approx. 28.9%) were identified by molecular diagnostic assays into sibling species. The An. gambiae s.l. complex was composed of Anopheles arabiensis (62.5%, n = 363/580), An. gambiae sensu stricto (An. gambiae s.s.; 0.7%, n = 4/580), Anopheles merus (0.7%, n = 4/580) and Anopheles quadriannulatus (0.2%, n = 1/580), with the remaining samples (35.5%, n = 206/580) unamplified. Anopheles funestus s.l. was composed of An. rivulorum (14.6%, n = 15/103) and An. leesoni (11.6%, n = 12/103); the remaining samples were unamplified (73.8%, n = 76/103). A total of 981 samples were subjected to PCR analysis for malaria parasite detection; of these 16 (1.6%) were confirmed to be positive for Plasmodium falciparum. The overall human blood index was 0.13 (32/238). Conclusions Anopheles gambiae, An. funestus and An. coustani are key malaria vectors in the Taveta region of Kenya, showing concurrent indoor and outdoor transmission. All of the vectors tested showed a higher propensity for bovine and goat blood than for human blood

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

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

A Phase 2b Randomised Trial of the Candidate Malaria Vaccines FP9 ME-TRAP and MVA ME-TRAP among Children in Kenya

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

Implementation of a structured paediatric admission record for district hospitals in Kenya – results of a pilot study

BACKGROUND: The structured admission form is an apparently simple measure to improve data quality. Poor motivation, lack of supervision, lack of resources and other factors are conceivably major barriers to their successful use in a Kenyan public hospital setting. Here we have examined the feasibility and acceptability of a structured paediatric admission record (PAR) for district hospitals as a means of improving documentation of illness. METHODS: The PAR was primarily based on symptoms and signs included in the Integrated Management of Childhood Illness (IMCI) diagnostic algorithms. It was introduced with a three-hour training session, repeated subsequently for those absent, aiming for complete coverage of admitting clinical staff. Data from consecutive records before (n = 163) and from a 60% random sample of dates after intervention (n = 705) were then collected to evaluate record quality. The post-intervention period was further divided into four 2-month blocks by open, feedback meetings for hospital staff on the uptake and completeness of the PAR. RESULTS: The frequency of use of the PAR increased from 50% in the first 2 months to 84% in the final 2 months, although there was significant variation in use among clinicians. The quality of documentation also improved considerably over time. For example documentation of skin turgor in cases of diarrhoea improved from 2% pre-intervention to 83% in the final 2 months of observation. Even in the area of preventive care documentation of immunization status improved from 1% of children before intervention to 21% in the final 2 months. CONCLUSION: The PAR was well accepted by most clinicians and greatly improved documentation of features recommended by IMCI for identifying and classifying severity of common diseases. The PAR could provide a useful platform for implementing standard referral care treatment guidelines