Combinatorial effects of malaria season, iron deficiency, and inflammation determine plasma hepcidin concentration in African children.

Hepcidin is the master regulatory hormone that governs iron homeostasis and has a role in innate immunity. Although hepcidin has been studied extensively in model systems, there is less information on hepcidin regulation in global health contexts where iron deficiency (ID), anemia, and high infectious burdens (including malaria) all coexist but fluctuate over time. We evaluated iron status, hepcidin levels, and determinants of hepcidin in 2 populations of rural children aged ≤8 years, in the Gambia and Kenya (total n = 848), at the start and end of a malaria season. Regression analyses and structural equation modeling demonstrated, for both populations, similar combinatorial effects of upregulating stimuli (iron stores and to a lesser extent inflammation) and downregulating stimuli (erythropoietic drive) on hepcidin levels. However, malaria season was also a significant factor and was associated with an altered balance of these opposing factors. Consistent with these changes, hepcidin levels were reduced whereas the prevalence of ID was increased at the end of the malaria season. More prevalent ID and lower hepcidin likely reflect an enhanced requirement for iron and an ability to efficiently absorb it at the end of the malaria season. These results, therefore, have implications for ID and malaria control programs.

Negative epistasis between the malaria-protective effects of alpha+-thalassemia and the sickle cell trait.

The hemoglobinopathies, disorders of hemoglobin structure and production, protect against death from malaria. In sub-Saharan Africa, two such conditions occur at particularly high frequencies: presence of the structural variant hemoglobin S and alpha(+)-thalassemia, a condition characterized by reduced production of the normal alpha-globin component of hemoglobin. Individually, each is protective against severe Plasmodium falciparum malaria, but little is known about their malaria-protective effects when inherited in combination. We investigated this question by studying a population on the coast of Kenya and found that the protection afforded by each condition inherited alone was lost when the two conditions were inherited together, to such a degree that the incidence of both uncomplicated and severe P. falciparum malaria was close to baseline in children heterozygous with respect to the mutation underlying the hemoglobin S variant and homozygous with respect to the mutation underlying alpha(+)-thalassemia. Negative epistasis could explain the failure of alpha(+)-thalassemia to reach fixation in any population in sub-Saharan Africa.

Effect of a fall in malaria transmission on morbidity and mortality in Kilifi, Kenya.

BACKGROUND: As efforts to control malaria are expanded across the world, understanding the role of transmission intensity in determining the burden of clinical malaria is crucial to the prediction and measurement of the effectiveness of interventions to reduce transmission. Furthermore, studies comparing several endemic sites led to speculation that as transmission decreases morbidity and mortality caused by severe malaria might increase. We aimed to assess the epidemiological characteristics of malaria in Kilifi, Kenya, during a period of decreasing transmission intensity. METHODS: We analyse 18 years (1990-2007) of surveillance data from a paediatric ward in a malaria-endemic region of Kenya. The hospital has a catchment area of 250 000 people. Clinical data and blood-film results for more than 61 000 admissions are reported. FINDINGS: Hospital admissions for malaria decreased from 18.43 per 1000 children in 2003 to 3.42 in 2007. Over 18 years of surveillance, the incidence of cerebral malaria initially increased; however, malaria mortality decreased overall because of a decrease in incidence of severe malarial anaemia since 1997 (4.75 to 0.37 per 1000 children) and improved survival among children admitted with non-severe malaria. Parasite prevalence, the mean age of children admitted with malaria, and the proportion of children with cerebral malaria began to change 10 years before hospitalisation for malaria started to fall. INTERPRETATION: Sustained reduction in exposure to infection leads to changes in mean age and presentation of disease similar to those described in multisite studies. Changes in transmission might not lead to immediate reductions in incidence of clinical disease. However, longitudinal data do not indicate that reductions in transmission intensity lead to transient increases in morbidity and mortality.

Acquisition of naturally occurring antibody responses to recombinant protein domains of Plasmodium falciparum erythrocyte membrane protein 1.

BACKGROUND: Antibodies targeting variant antigens expressed on the surface of Plasmodium falciparum infected erythrocytes have been associated with protection from clinical malaria. The precise target for these antibodies is unknown. The best characterized and most likely target is the erythrocyte surface-expressed variant protein family Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). METHODS: Using recombinant proteins corresponding to five domains of the expressed A4 var gene, A4 PfEMP1, the naturally occurring antibody response was assessed, by ELISA, to each domain in serum samples obtained from individuals resident in two communities of differing malaria transmission intensity on the Kenyan coast. Using flow cytometry, the correlation in individual responses to each domain with responses to intact A4-infected erythrocytes expressing A4 PfEMP1 on their surface as well as responses to two alternative parasite clones and one clinical isolate was assessed. RESULTS: Marked variability in the prevalence of responses between each domain and between each transmission area was observed, as wasa strong correlation between age and reactivity with some but not all domains. Individual responses to each domain varied strikingly, with some individuals showing reactivity to all domains and others with no reactivity to any, this was apparent at all age groups. Evidence for possible cross-reactivity in responses to the domain DBL4gamma was found. CONCLUSION: Individuals acquire antibodies to surface expressed domains of a highly variant protein. The finding of potential cross-reactivity in responses to one of these domains is an important initial finding in the consideration of potential vaccine targets.

Prevalence and correlates of home delivery amongst HIV-infected women attending care at a rural public health facility in Coastal Kenya.

BACKGROUND: Home delivery, referring to pregnant women giving birth in the absence of a skilled birth attendant, is a significant contributor to maternal mortality, and is encouragingly reported to be on a decline in the general population in resource limited settings. However, much less is known about home delivery amongst HIV-infected women in sub-Saharan Africa (sSA). We described the prevalence and correlates of home delivery among HIV-infected women attending care at a rural public health facility in Kilifi, Coastal Kenya. METHODS: A cross-sectional design using mixed methods was used. Quantitative data were collected using interviewer-administered questionnaires from HIV-infected women with a recent pregnancy (within 5 years, n = 425), whilst qualitative data were collected using focused group discussions (FGD, n = 5). Data were analysed using logistic regression and a thematic framework approach respectively. RESULTS: Overall, 108 (25.4%, [95% CI: 21.3-29.8]) participants delivered at home. Correlates of home delivery included lack of formal education (aOR 12.4 [95% CI: 3.4-46.0], p<0.001), history of a previous home delivery (2.7 [95% CI:1.2-6.0], p = 0.019) and being on highly active antiretroviral therapy (HAART, 0.4 [95% CI:0.2-0.8], p = 0.006).Despite a strong endorsement against home delivery, major thematic challenges included consumer-associated barriers, health care provider associated barriers and structural barriers. CONCLUSION: A quarter of HIV-infected women delivered at home, which is comparable to estimates reported from the general population in this rural setting, and much lower than estimates from other sSA settings. A tailored package of care targeting women with no formal education and with a history of a previous home delivery, coupled with interventions towards scaling up HAART and improving the quality of maternal care in HIV-infected women may positively contribute to a decline in home delivery and subsequent maternal mortality in this setting.

The haptoglobin 2-2 genotype is associated with a reduced incidence of Plasmodium falciparum malaria in children on the coast of Kenya.

BACKGROUND: Haptoglobin (Hp) genotype determines the efficiency of hemoglobin clearance after malaria-induced hemolysis and alters antioxidant and immune functions. The Hp2 allele is thought to have spread under strong selection pressure, but it is unclear whether this is due to protection from malaria or other diseases. METHODS: We monitored the incidence of febrile malaria and other childhood illnesses with regard to Hp genotype in a prospective cohort of 312 Kenyan children during 558.3 child-years of follow-up. We also conducted 7 cross-sectional surveys to determine the prevalence of Plasmodium falciparum parasitemia. RESULTS: The Hp2/2 genotype was associated with a 30% reduction in clinical malarial episodes (adjusted incidence rate ratio, 0.67; P=.008 for Hp2/2 vs. Hp1/1 and Hp2/1 combined). Protection increased with age; there was no protection in the first 2 years of life, 30% protection at > or = 2 years of age, and 50% protection from 4-10 years of age. Children with the Hp1/1 genotype had a significantly lower rate of nonmalarial fever (P=.001). CONCLUSIONS: Balancing selection pressures may have influenced the spread of the Hp gene. Our observations suggest that the Hp2 allele may have spread as a result of protection from malaria, and the Hp1 allele may be sustained by protection from other infections.

The effect of alpha+-thalassaemia on the incidence of malaria and other diseases in children living on the coast of Kenya.

BACKGROUND: The alpha-thalassaemias are the commonest genetic disorders of humans. It is generally believed that this high frequency reflects selection through a survival advantage against death from malaria; nevertheless, the epidemiological description of the relationships between alpha-thalassaemia, malaria, and other common causes of child mortality remains incomplete. METHODS AND FINDINGS: We studied the alpha+-thalassaemia-specific incidence of malaria and other common childhood diseases in two cohorts of children living on the coast of Kenya. We found no associations between alpha+-thalassaemia and the prevalence of symptomless Plasmodium falciparum parasitaemia, the incidence of uncomplicated P. falciparum disease, or parasite densities during mild or severe malaria episodes. However, we found significant negative associations between alpha+-thalassaemia and the incidence rates of severe malaria and severe anaemia (haemoglobin concentration < 50 g/l). The strongest associations were for severe malaria anaemia (> 10,000 P. falciparum parasites/mul) and severe nonmalaria anaemia; the incidence rate ratios and 95% confidence intervals (CIs) for alpha+-thalassaemia heterozygotes and homozygotes combined compared to normal children were, for severe malaria anaemia, 0.33 (95% CI, 0.15,0.73; p = 0.006), and for severe nonmalaria anaemia, 0.26 (95% CI, 0.09,0.77; p = 0.015). CONCLUSIONS: Our observations suggest, first that selection for alpha+-thalassaemia might be mediated by a specific effect against severe anaemia, an observation that may lead to fresh insights into the aetiology of this important condition. Second, although alpha+-thalassaemia is strongly protective against severe and fatal malaria, its effects are not detectable at the level of any other malaria outcome; this result provides a cautionary example for studies aimed at testing malaria interventions or identifying new malaria-protective genes.

Contrasting signatures of selection on the Plasmodium falciparum erythrocyte binding antigen gene family.

Erythrocyte binding antigens of Plasmodium falciparum are involved in erythrocyte invasion, and may be targets of acquired immunity. Of the five eba genes, protein products have been detected for eba-175, eba-181 and eba-140, but not for psieba-165 or ebl-1, providing opportunity for comparative analysis of genetic variation to identify selection. Region II of each of these genes was sequenced from a cross-sectional sample of parasites in an endemic Kenyan population, and the frequency distributions of polymorphisms analysed. A positive value of Tajima's D was observed for eba-175 (D=1.13) indicating an excess of intermediate frequency polymorphisms, while all other genes had negative values, the most negative being ebl-1 (D=-2.35) followed by psieba-165 (D=-1.79). The eba-175 and ebl-1 genes were then studied in a sample of parasites from Thailand, for which a positive Tajima's D value was again observed for eba-175 (D=1.79), and a negative value for ebl-1 (D=-1.85). This indicates that eba-175 is under balancing selection in each population, in strong contrast to the other members of the gene family, particularly ebl-1 and psieba-165 that may have been under recent directional selection. Population expansion simulations were performed under a neutral model, further supporting the departures from neutrality of these genes.

A polymorphism of intercellular adhesion molecule-1 is associated with a reduced incidence of nonmalarial febrile illness in Kenyan children.

An intercellular adhesion molecule-1 polymorphism (ICAM-1(Kilifi)) is present at a high frequency across sub-Saharan Africa, and its presence may increase susceptibility to cerebral malaria. Here, we report that, compared with children in whom wild-type intercellular adhesion molecule-1 is present, the incidence of nonmalarial fever is significantly lower among those homozygous for ICAM-1(Kilifi). We propose that ICAM-1(Kilifi) may be associated with reduced rates of tissue damage and of death due to sepsis.

Heritability of malaria in Africa.

BACKGROUND: While many individual genes have been identified that confer protection against malaria, the overall impact of host genetics on malarial risk remains unknown. METHODS AND FINDINGS: We have used pedigree-based genetic variance component analysis to determine the relative contributions of genetic and other factors to the variability in incidence of malaria and other infectious diseases in two cohorts of children living on the coast of Kenya. In the first, we monitored the incidence of mild clinical malaria and other febrile diseases through active surveillance of 640 children 10 y old or younger, living in 77 different households for an average of 2.7 y. In the second, we recorded hospital admissions with malaria and other infectious diseases in a birth cohort of 2,914 children for an average of 4.1 y. Mean annual incidence rates for mild and hospital-admitted malaria were 1.6 and 0.054 episodes per person per year, respectively. Twenty-four percent and 25% of the total variation in these outcomes was explained by additively acting host genes, and household explained a further 29% and 14%, respectively. The haemoglobin S gene explained only 2% of the total variation. For nonmalarial infections, additive genetics explained 39% and 13% of the variability in fevers and hospital-admitted infections, while household explained a further 9% and 30%, respectively. CONCLUSION: Genetic and unidentified household factors each accounted for around one quarter of the total variability in malaria incidence in our study population. The genetic effect was well beyond that explained by the anticipated effects of the haemoglobinopathies alone, suggesting the existence of many protective genes, each individually resulting in small population effects. While studying these genes may well provide insights into pathogenesis and resistance in human malaria, identifying and tackling the household effects must be the more efficient route to reducing the burden of disease in malaria-endemic areas.

An immune basis for malaria protection by the sickle cell trait.

BACKGROUND: Malaria resistance by the sickle cell trait (genotype HbAS) has served as the prime example of genetic selection for over half a century. Nevertheless, the mechanism of this resistance remains the subject of considerable debate. While it probably involves innate factors such as the reduced ability of Plasmodium falciparum parasites to grow and multiply in HbAS erythrocytes, recent observations suggest that it might also involve the accelerated acquisition of malaria-specific immunity. METHODS AND FINDINGS: We studied the age-specific protection afforded by HbAS against clinical malaria in children living on the coast of Kenya. We found that protection increased with age from only 20% in the first 2 y of life to a maximum of 56% by the age of 10 y, returning thereafter to 30% in participants greater than 10 y old. CONCLUSIONS: Our observations suggest that malaria protection by HbAS involves the enhancement of not only innate but also of acquired immunity to the parasite. A better understanding of the underlying mechanisms might yield important insights into both these processes.

The effects of untreated bednets on malaria infection and morbidity on the Kenyan coast.

A study was conducted in order to determine whether children that slept under untreated bednets were protected against both malaria infection and clinical disease compared with children not sleeping under bednets. The study was conducted in Kilifi District, Kenya, during the malaria season (June-August, 2000) and involved 416 children aged < or = 10 years. Data collected from a cross-sectional survey showed evidence of protection against malaria infection among children sleeping under untreated bednets in good condition compared with those not using nets (adjusted odds ratio [AOR] = 0.4, 95% CI 0.22-0.72, P = 0.002). There was no evidence of a protective effect against infection when comparing those that used untreated bednets that were worn and those not using nets (AOR = 0.75, 95% CI 0.34-1.63, P = 0.47). When these same children were followed-up during the malaria season, there was evidence of a lower rate of clinical malaria among those that used untreated nets in good condition (adjusted incidence rate ratio = 0.65, 95% CI 0.45-0.94, P = 0.022), while the rate of clinical malaria among those that used untreated bednets that were worn was similar to that of those that did not use bednets. In the face of persistent failure of communities to take up net retreatment, there is hope that untreated nets will offer some protection against malaria infection and disease compared with not using nets at all.

Transmission-dependent tolerance to multiclonal Plasmodium falciparum infection.

Whether the number of concurrent clones in asymptomatic Plasmodium falciparum infections reflects the degree of host protection was investigated in children living in areas with different levels of transmission on the coast of Kenya. The number of concurrent clones was determined on the basis of polymorphism in msp2, which encodes the vaccine candidate antigen merozoite surface protein 2. In a low-transmission area, most children had monoclonal infections, and diversity did not predict a risk of clinical malaria. In an area of moderate transmission, asymptomatic infections with 2 clones were, compared with 1 clone, associated with an increased risk of subsequent malaria. In a comparative assessment in a high-transmission area in Tanzania, multiclonal infections conferred a reduced risk. The different nonlinear associations between the number of clones and malaria morbidity suggest that levels of tolerance to multiclonal infections are transmission dependent as a result of cumulative exposure to antigenically diverse P. falciparum infections.

Helminth infection and eosinophilia and the risk of Plasmodium falciparum malaria in 1- to 6-year-old children in a malaria endemic area.

BACKGROUND: Helminth infection is common in malaria endemic areas, and an interaction between the two would be of considerable public health importance. Animal models suggest that helminth infections may increase susceptibility to malaria, but epidemiological data has been limited and contradictory. METHODOLOGY/PRINCIPAL FINDINGS: In a vaccine trial, we studied 387 one- to six-year-old children for the effect of helminth infections on febrile Plasmodium falciparum malaria episodes. Gastrointestinal helminth infection and eosinophilia were prevalent (25% and 50% respectively), but did not influence susceptibility to malaria. Hazard ratios were 1 for gastrointestinal helminth infection (95% CI 0.6-1.6) and 0.85 and 0.85 for mild and marked eosinophilia, respectively (95% CI 0.56-1.76 and 0.69-1.96). Incident rate ratios for multiple episodes were 0.83 for gastro-intestinal helminth infection (95% CI 0.5-1.33) and 0.86 and 0.98 for mild and marked eosinophilia (95% CI 0.5-1.4 and 0.6-1.5). CONCLUSIONS/SIGNIFICANCE: There was no evidence that infection with gastrointestinal helminths or urinary schistosomiasis increased susceptibility to Plasmodium falciparum malaria in this study. Larger studies including populations with a greater prevalence of helminth infection should be undertaken.

Relationship between exposure, clinical malaria, and age in an area of changing transmission intensity.

The relationship between malaria transmission intensity and clinical disease is important for predicting the outcome of control measures that reduce transmission. Comparisons of hospital data between areas of differing transmission intensity suggest that the mean age of hospitalized clinical malaria is higher under relatively lower transmission, but the total number of episodes is similar until transmission drops below a threshold, where the risks of hospitalized malaria decline. These observations have rarely been examined longitudinally in a single community where transmission declines over time. We reconstructed 16 years (1991-2006) of pediatric hospital surveillance data and infection prevalence surveys from a circumscribed geographic area on the Kenyan coast. The incidence of clinical malaria remained high, despite sustained reductions in exposure to infection. However, the age group experiencing the clinical attacks of malaria increased steadily as exposure declined and may precede changes in the number of episodes in an area with declining transmission.

The induction and persistence of T cell IFN-gamma responses after vaccination or natural exposure is suppressed by Plasmodium falciparum.

Epidemiological observations suggest that T cell immunity may be suppressed in malaria-endemic areas. In vitro studies, animal models, and limited data in humans link immunosuppression with malaria, malnutrition, and other parasitic infections. However, there are no data to determine whether malaria-induced immunosuppression is significant in the long-term, or relative data comparing it with other factors in malaria-endemic areas, so as to measure the impact of malaria, other parasitic disease, nutritional status, age. and location on the acquisition and longevity of IFN-gamma responses in children in Kenya. We studied these factors in two cohorts of 1- to 6-year-old children in a malaria-endemic area. T cell responses were induced by vaccination in one cohort, and acquired as a result of natural exposure in a second cohort. Serial ELISPOT assays conducted over a 1-year period measured the induction and kinetics of IFN-gamma production in response to the malaria Ag thrombospondin-related adhesion protein. Induced responses in both cohorts and the longevity of response in the vaccinated cohort were fitted to potential explanatory variables. Parasitemia was prospectively associated with reduced IFN-gamma-producing T cells in both cohorts (by 15-25%), and both parasitemia and episodes of febrile malaria were associated with 19 and 31% greater attrition of T cell responses, respectively. Malaria may reduce the efficacy vaccinations such as bacillus Calmette-Guérin and investigational T cell-inducing vaccines, and may delay the acquisition of immunity following natural exposure to malaria and other pathogens.

High levels of serum antibodies to merozoite surface protein 2 of Plasmodium falciparum are associated with reduced risk of clinical malaria in coastal Kenya.

The merozoite surface protein (MSP) 2 is a vaccine candidate antigen of Plasmodium falciparum that is polymorphic in natural populations. In a prospective cohort study in two coastal populations of Kenya using recombinant proteins derived from the two major allelic types of MSP2, high serum levels of IgG to MSP2 were associated with protection from clinical malaria. This protection was independent of that associated with antibodies to another vaccine candidate antigen (AMA1) in these populations. However, low antibody levels to MSP2 appeared to be associated with increased susceptibility to malaria within people who were parasite negative at the time of serum collection. These data suggest that an MSP2 based vaccine should be designed to induce high level antibody responses against the different MSP2 types present globally in P. falciparum populations and that MSP2 could be combined with other P. falciparum antigens to form a multi-component malaria vaccine.

Case definitions of clinical malaria under different transmission conditions in Kilifi District, Kenya.

BACKGROUND: Clear case definitions of malaria are an essential means of evaluating the effectiveness of present and proposed interventions in malaria. The clinical signs of malaria are nonspecific, and parasitemia accompanied by a fever may not be sufficient to define an episode of clinical malaria in endemic areas. We defined and quantified cases of malaria in people of different age groups from 2 areas with different rates of transmission of malaria. METHODS: A total of 1602 people were followed up weekly for 2 years, and all the cases of fever accompanied by parasitemia were identified. Logistic regression methods were used to derive case definitions of malaria. RESULTS: Two case definitions of malaria were derived: 1 for children 1-14 years old and 1 for infants (<1 year old) and older children and adults (> or =15 years old). We also found a higher number of episodes of clinical malaria per person per year in people from an area of low transmission of malaria, compared with the number of episodes in those from an area of higher transmission (0.84 vs. 0.55 episodes/person/year; incidence rate ratio, 0.66 [95% confidence interval, 0.61-0.72]; P<.001). CONCLUSIONS: Case definitions of malaria are bound to be altered by factors that affect immunity, such as age and transmission. Case definitions may, however, be affected by other immunity-altering factors, such as HIV and vaccination status, and this needs to be borne in mind during vaccine trials.

Clinical algorithms for malaria diagnosis lack utility among people of different age groups.

We conducted a study to determine whether clinical algorithms would be useful in malaria diagnosis among people living in an area of moderate malaria transmission within Kilifi District in Kenya. A total of 1602 people of all age groups participated. We took smears and recorded clinical signs and symptoms (prompted or spontaneous) of all those presenting to the study clinic with a history of fever. A malaria case was defined as a person presenting to the clinic with a history of fever and concurrent parasitaemia. A set of clinical signs and symptoms (algorithms) with the highest sensitivity and specificity for diagnosing a malaria case was selected for the age groups /=15 years. These age-optimized derived algorithms were able to identify about 66% of the cases among those <15 years of age but only 23% of cases among adults. Were these algorithms to be used as a basis for a decision on treatment among those presenting to the clinic, 16% of children /=5000 parasites/microl of blood would be sent home without treatment. Clinical algorithms therefore appear to have little utility in malaria diagnosis, performing even worse in the older age groups, where avoiding unnecessary use of anti-malarials would make more drugs available to the really needy population of children under 5 years of age.

Sickle cell trait and the risk of Plasmodium falciparum malaria and other childhood diseases.

BACKGROUND: The gene for sickle hemoglobin (HbS) is a prime example of natural selection. It is generally believed that its current prevalence in many tropical populations reflects selection for the carrier form (sickle cell trait [HbAS]) through a survival advantage against death from malaria. Nevertheless, >50 years after this hypothesis was first proposed, the epidemiological description of the relationships between HbAS, malaria, and other common causes of child mortality remains incomplete. METHODS: We studied the incidence of falciparum malaria and other childhood diseases in 2 cohorts of children living on the coast of Kenya. RESULTS: The protective effect of HbAS was remarkably specific for falciparum malaria, having no significant impact on any other disease. HbAS had no effect on the prevalence of symptomless parasitemia but was 50% protective against mild clinical malaria, 75% protective against admission to the hospital for malaria, and almost 90% protective against severe or complicated malaria. The effect of HbAS on episodes of clinical malaria was mirrored in its effect on parasite densities during such episodes. CONCLUSIONS: The present data are useful in that they confirm the mechanisms by which HbAS confers protection against malaria and shed light on the relationships between HbAS, malaria, and other childhood diseases.