NFKBIZ polymorphisms and susceptibility to pneumococcal disease in European and African populations.

The proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) has a central role in host defence against pneumococcal disease. Both rare mutations and common polymorphisms in the NFKBIA gene encoding the NF-kappaB inhibitor, IkappaB-alpha, associate with susceptibility to bacterial disease, but the possible role of polymorphisms within the related IkappaB-zeta gene NFKBIZ in the development of invasive pneumococcal disease (IPD) has not been reported previously. To investigate this further, we examined the frequencies of 22 single-nucleotide polymorphisms spanning NFKBIZ in two case-control studies, comprising UK Caucasian (n=1008) and Kenyan (n=723) individuals. Nine polymorphisms within a single UK linkage disequilibrium (LD) block and all four polymorphisms within the equivalent, shorter Kenyan LD block displayed either a significant association with IPD or a trend towards association. For each polymorphism, heterozygosity was associated with protection from IPD when compared with the combined homozygous states (for example, for rs600718, Mantel-Haenszel 2 x 2 chi(2)=7.576, P=0.006, odds ratio (OR)=0.67, 95% confidence interval (95% CI) for OR: 0.51-0.88; for rs616597, Mantel-Haenszel 2 x 2 chi(2)=8.715, P=0.003, OR=0.65, 95% CI: 0.49-0.86). We conclude that multiple NFKBIZ polymorphisms associate with susceptibility to IPD in humans. The study of multiple populations may aid in fine mapping of associations within extensive regions of strong LD ('transethnic mapping').

Variation in the ICAM1 gene is not associated with severe malaria phenotypes.

Evidence from autopsy and in vitro binding studies suggests that adhesion of erythrocytes infected with Plasmodium falciparum to the human host intercellular adhesion molecule (ICAM)-1 receptor is important in the pathogenesis of severe malaria. Previous association studies between polymorphisms in the ICAM1 gene and susceptibility to severe malarial phenotypes have been inconclusive and often contradictory. We performed genetic association studies with 15 single nucleotide polymorphisms (SNPs) around the ICAM1 locus. All SNPs were screened in a family study of 1071 trios from The Gambia, Malawi and Kenya. Two key non-synonymous SNPs with previously reported associations, rs5491 (K56M or 'ICAM-1(Kilifi)') and rs5498 (K469E), were tested in an additional 708 Gambian trios and a case-control study of 4058 individuals. None of the polymorphisms were associated with severe malaria phenotypes. Pooled results across our studies for ICAM-1(Kilifi) were, in severe malaria, odds ratio (OR) 1.02, 95% confidence interval (CI) 0.96-1.09, P=0.54, and cerebral malaria OR 1.07, CI 0.97-1.17, P=0.17. We assess the available epidemiological, population genetic and functional evidence that links ICAM-1(Kilifi) to severe malaria susceptibility.

Positive replication and linkage disequilibrium mapping of the chromosome 21q22.1 malaria susceptibility locus.

Four cytokine receptor genes are located on Chr21q22.11, encoding the alpha and beta subunits of the interferon-alpha receptor (IFNAR1 and IFNAR2), the beta subunit of the interleukin 10 receptor (IL10RB) and the second subunit of the interferon-gamma receptor (IFNGR2). We previously reported that two variants in IFNAR1 were associated with susceptibility to malaria in Gambians. We now present an extensive fine-scale mapping of the associated region utilizing 45 additional genetic markers obtained from public databases and by sequencing a 44 kb region in and around the IFNAR1 gene in 24 Gambian children (12 cases/12 controls). Within the IFNAR1 gene, a newly studied C --> G single-nucleotide polymorphism (IFNAR1 272354c-g) at position -576 relative to the transcription start was found to be more strongly associated with susceptibility to severe malaria. Association was observed in three populations: in Gambian (P=0.002), Kenyan (P=0.022) and Vietnamese (P=0.005) case-control studies. When all three studies were combined, using the Mantel-Haenszel test, the presence of IFNAR1 -576G was associated with a substantially elevated risk of severe malaria (N=2444, OR=1.38, 95% CI: 1.17-1.64; P=1.7 x 10(-4)). This study builds on previous work to further highlight the importance of the type-I interferon pathway in malaria susceptibility and illustrates the utility of typing SNPs within regions of high linkage disequilibrium in multiple populations to confirm initial positive associations.

Elevated plasma urokinase receptor predicts low birth weight in maternal malaria.

The blood level of soluble urokinase receptor (suPAR) is increased and associated with a poor clinical or fatal outcome in children with acute malaria. This study hypothesized that the suPAR level would be associated with foetal outcome in maternal malaria. suPAR was measured by ELISA in maternal and cord plasma samples taken during delivery in 253 pregnant Kenyan women stratified according to placental histology: no malaria infection (non-infected), active or active-chronic infection (actively infected) or past-chronic infection (past-infected). Maternal-suPAR was higher in actively infected women (median 3.93 (IQR 2.92-5.29) ng/mL) compared with non-infected (median 2.78 (IQR 1.86-3.87) ng/mL, P = 0.001) and past-infected (median 2.67 (IQR 1.94-3.7) ng/mL, P = 0.012) women. Cord-suPAR was comparable across the groups (median 2.98 (IQR 2.38-3.77) ng/mL). In actively infected women, maternal-suPAR and gestational age were the only independent predictors of birth weight in multivariate linear regression adjusted for maternal-suPAR, HIV-1 infection, age, BMI, haemoglobin, peripheral parasitaemia, parity and gestational age; 1 ng/mL higher maternal-suPAR predicted -56 g (95% CI -100 to -12, P = 0.016) reduced birth weight. Cord-suPAR could not predict birth weight after adjusting for gestational age. Future studies are warranted to investigate whether the maternal suPAR level is increased earlier in pregnancy in women with active placental malaria infection and whether early maternal suPAR measurements can predict birth weight. If so, measurements of maternal suPAR early in pregnancy might then potentially identify women with increased needs for antenatal care and intervention.

Trust and informed consent: insights from community members on the Kenyan coast.

Trust is an important theme running through the literature on the ethics of biomedical research, but it is rarely given centre stage. In this paper, we present data gathered from a study aimed at exploring community views regarding the informed consent processes carried out by a large research centre on the Kenyan Coast. The findings point to the centrality of trust and elements of mistrust in general community views, in parents' (mis)understanding of studies they consent their children to be involved in, in refusals and concerns, and in community members' views about whether informed consent is a relevant and practical model to follow. Tentative ideas on how trust and a healthy mistrust might be balanced highlight the importance of strengthening communication surrounding basic health care as well as research, and of fostering 'an inner generated ethic of service'. The latter is particularly fundamental, but cannot be built and regulated through the laws, policies and guidelines that currently govern biomedical research practice.

'Even if they ask you to stand by a tree all day, you will have to do it (laughter)...!': community voices on the notion and practice of informed consent for biomedical research in developing countries.

Ethical dilemmas in biomedical research, especially in vulnerable populations, often spark heated debate. Despite recommendations and guidelines, many issues remain controversial, including the relevance, prioritisation and application of individual voluntary informed consent in non-Western settings. The voices of the people likely to be the subjects of research have been notably absent from the debate. We held discussions with groups of community members living in the rural study area of a large research unit in Kenya. Discussions were facilitated by three research study vignettes outlining one field-based and two hospital-based studies being planned or taking place at the time. In addition to gathering general views about the aims and activities of the research unit, questions focused on whether consent should be sought for studies, and if so from whom (chiefs, elders, men/women, children), and on ascertaining whether there are any special concerns about the physical act of signing consent forms. The findings revealed the community's difficulty in distinguishing research from clinical investigations conducted in clinical settings. There was a spectrum of views regarding perceived appropriateness of consent procedures, in part because of difficulty in disentangling clinical from research aims, and because of other challenges to applying consent in practice. Debates between community members highlight the inadequacy of simplistic assumptions about community members' views on informed consent, and the complexity of incorporating lay opinions into biomedical research. Failure to appreciate these issues risks exaggerating differences between settings, and underestimating the time and resources required to ensure meaningful community involvement in research processes. Ultimately, it risks inadequately responding to the needs and values of those on whom the success of most biomedical research depends. Although compliance with community views does not necessarily make the research more ethical, it is argued that community opinions on local issues and practices should inform ethical decision-making in health research.

Pre-transfusion management of children with severe malarial anaemia: a randomised controlled trial of intravascular volume expansion.

Symptomatic severe malarial anaemia (SMA) has a high fatality rate of 30-40%; most deaths occur in children awaiting blood transfusion. Blood transfusion services in most of Africa are not capable of delivering adequate supplies of safe blood in a timely manner to critically ill children with SMA. Contrary to widely held belief, hypovolaemia, rather than heart failure, has emerged as a common complication in such children. We examined the safety of pre-transfusion management (PTM) by volume expansion, aimed at stabilizing children and obviating the urgency for blood transfusion. Kenyan children with severe falciparum anaemia (haemoglobin <5 g/dl) and respiratory distress were randomly assigned to 20 ml/kg of 4.5% albumin or 0.9% saline or maintenance only (control) while awaiting blood transfusion. PTM was apparently safe since it did not lead to the development of pulmonary oedema or other adverse events. There was no significant difference in the primary outcome [mean percentage reduction in base excess between admission and 8 h (95% confidence interval)] between the control group 42% (19-66%) albumin group 44% (32-57%) and saline group 36% (16-57%); adjusted analysis of variance F=0.31, P=0.7. However, the number of children requiring emergency interventions was significantly greater in the control group, four of 18 (22%) than the saline group 0 of 20 (P=0.03). We have established the safety of this PTM in children with SMA whilst awaiting blood transfusion at a hospital with an adequate blood-banking program. The impact on mortality should be assessed where blood transfusion services are unable to supply emergency transfusions.

Understanding of informed consent in a low-income setting: three case studies from the Kenyan Coast.

In our research unit on the Kenyan Coast, parents sign consent for over 4000 children to be involved in research activities every year. Children are recruited into studies ranging from purely observational research to the testing of new procedures and drugs. Thousands more community members consent verbally or in writing to the interviews and sometimes invasive procedures required in community-based research. Although every study and consent form is reviewed in advance by independent national and international committees, the views and understanding of the 'subjects' of these activities had not been documented before this study. In this paper, we focus on participant understanding of one field-based and two hospital-based studies, all of which involve blood sampling. The findings highlight a range of inter-related issues for consideration in the study setting and beyond, including conceptual and linguistic barriers to communicating effectively about research, the critical and complex role of communicators (fieldworkers and nurses) in consent procedures, features of research unit-community relations which impact on these processes, and the special sensitivity of certain issues such as blood sampling. These themes and emerging recommendations are expected to be relevant to, and would benefit from, experiences and insights of researchers working elsewhere.

Severe P. falciparum malaria in Kenyan children: evidence for hypovolaemia.

BACKGROUND: The role of volume resuscitation in severe Plasmodium falciparum malaria is controversial. AIM: To examine the role of hypovolaemia in severe childhood malaria. STUDY DESIGN: Retrospective review. METHODS: We studied 515 children admitted with severe malaria to a high-dependency unit (HDU) in Kilifi, Kenya. On admission to the HDU, children underwent a further assessment of vital signs and a standard clinical examination. RESULTS: Factors associated with a fatal outcome included deep breathing or acidosis (base excess below -8), hypotension (systolic blood pressure <80 mmHg), raised plasma creatinine (>80 micro mol/l), low oxygen saturation (<90%), dehydration and hypoglycaemia (<2.5 mmol/l). Shock was present in 212/372 (57%) children, of whom 37 (17.5%) died, and was absent in 160, of who only 7 (4.4%) died (chi(2) = 14.9; p = 0.001). DISCUSSION: Impaired tissue perfusion may play a role in the mortality of severe malaria. Moreover, volume resuscitation, an important life-saving intervention in children with hypovolaemia, should be considered in severe malaria with evidence of impaired tissue perfusion.

A promoter polymorphism in the gene encoding interleukin-12 p40 (IL12B) is associated with mortality from cerebral malaria and with reduced nitric oxide production.

Interleukin-12 (IL-12) is an important regulatory cytokine in infection and immunity. Administration of IL-12 may reduce complications of severe malaria in rodents. Polymorphisms in IL12B, the gene encoding the IL-12 p40 subunit, influence the secretion of IL-12 and susceptibility to Type 1 diabetes. We therefore investigated whether IL12B polymorphisms may affect the outcome of severe malaria. Homozygosity for a polymorphism in the IL12B promoter was associated with increased mortality in Tanzanian children having cerebral malaria but not in Kenyan children with severe malaria. Furthermore, homozygotes for the IL12B promotor polymorphism had decreased production of nitric oxide, which is in part regulated by IL-12 activity. These studies suggest that IL12B polymorphisms, via regulation of IL-12 production, may influence the outcome of malaria infection in at least one African population.

Impaired uteroplacental blood flow in pregnancies complicated by falciparum malaria.

OBJECTIVE: In endemic areas, maternal malaria infection is usually asymptomatic. However, it is known that infected maternal erythrocytes sequester in the intervillous space of the placenta. There is a strong association between placental malaria infection and both low birth weight (LBW) and severe maternal anemia. We aimed to determine whether impaired uteroplacental blood flow might account for the low infant birth weight associated with maternal falciparum malaria infection. METHODS: This observational study was carried out during a large double-blind, randomized, controlled trial of an antimalarial drug intervention for primigravidae. Nine hundred and ninety-five women were recruited from the antenatal clinic at a district hospital on the Kenya coast and had at least one Doppler ultrasound scan. Uterine artery resistance index and the presence or absence of a diastolic notch were recorded. In the third trimester, blood was taken for hemoglobin and malaria film. RESULTS: Malaria infection at 32-35 weeks of gestation was associated with abnormal uterine artery flow velocity waveforms on the day of blood testing (relative risk (RR) 2.11, 95% confidence interval (CI) 1.24-3.59, P = 0.006). This association persisted after controlling for pre-eclampsia. Impaired uteroplacental blood flow in the women studied was also predictive of poor perinatal outcome, including low birth weight, preterm delivery and perinatal death. The risk of preterm delivery in women with histological evidence of past placental malaria infection was more than twice that of women without infection (RR 2.33, 95% CI 1.31-4.13, P = 0.004). CONCLUSIONS: Uteroplacental hemodynamics are altered in the presence of maternal falciparum malaria infection. This may account for some of the excess of LBW babies observed in malaria endemic areas. Strategies that prevent or clear placental malaria may confer perinatal benefit through preservation of placental function.

Malaria in pregnancy: adverse effects on haemoglobin levels and birthweight in primigravidae and multigravidae.

BACKGROUND: In areas of endemic transmission, malaria in pregnancy is associated with severe maternal anaemia and low birthweight babies. The prevalence of infection is highest in primigravidae (PG), and hence control efforts are usually geared towards this high risk group. Using a sensitive measure of placental infection, we investigated the relationship between active-acute, active-chronic and past placental infection with maternal anaemia and low birthweight in women of all gravidities. METHODS: Between January 1996 and July 1997, 912 women delivering in Kilifi District Hospital, Kenya, were recruited. Haemoglobin and peripheral malaria slides were taken prior to delivery, placental biopsies and smears were taken at the time of delivery and birthweight and maternal height and weight were measured soon after birth. Information was obtained on socio-economic and educational status. The association between placental malaria, severe anaemia and low birthweight was investigated for women of different gravidities. FINDINGS: By placental histology, the prevalence of active or past malaria in all gravidities was high, ranging from 64% in PG to 30% in gravidities 5 and above. In gravidities 1-4, active malaria infection was associated with severe maternal anaemia, adjusted OR 2.21 (95% CI 1.36, 3.61). There was a significant interaction between chronic or past malaria and severe anaemia in their effects on birthweight, whereby the risk of low birthweight was very high in women with both chronic or past placental malaria and severe anaemia: OR 4.53 (1.19, 17.2) in PG; 13.5 (4.57, 40) in gravidities 2-4. INTERPRETATION: In this area of moderate malaria transmission, women of all parities have substantially increased risk of low birthweight and severe anaemia as a result of malaria infection in pregnancy. The risk of low birthweight is likely to be particularly high in areas with a high prevalence of severe anaemia.

Screening for severe anaemia in pregnancy in Kenya, using pallor examination and self-reported morbidity.

Severe anaemia in pregnancy is an important preventable cause of maternal and perinatal morbidity and mortality. Different methods of screening for severe anaemia in pregnancy were evaluated in a 2-phased study conducted in Kilifi, Kenya. In phase 1 (in 1994/95), pallor testing was evaluated alone and in addition to raised respiratory/pulse rates: 1787 pregnant women were examined by one of 2 midwives. Sensitivities for detecting severe anaemia (haemoglobin < 7 g/dL) were 62% and 69% and specificities 87% and 77%, respectively for each of the midwives. Addition of high pulse rate increased sensitivity to 77% and 81%, but specificity reduced to 60% and 51%, respectively. In phase 2, following qualitative in-depth work, a screening questionnaire was developed. An algorithm based on screening questions had 80% sensitivity and 40% specificity. Midwife pallor-assessment was conducted following the screening questionnaire. In this phase (conducted in 1997), the midwife performed very highly in detecting severe anaemia, achieving sensitivity of 84% and specificity of 92%. Spending a few minutes asking women questions may have improved the ability to interpret pallor findings. This study demonstrates the value of pallor testing and raises alternative approaches to improving it.

The epidemiology of hookworm infection and its contribution to anaemia among pre-school children on the Kenyan coast.

Intestinal nematode infections are recognized as a major public health problem, and helminth control is currently being directed towards school-aged children who are known to harbour the heaviest infections and are most likely to suffer from associated morbidity. However, few data are available for the epidemiology of intestinal nematodes in pre-school children in Africa, and the contribution of hookworm infection to the aetiology and severity of anaemia among pre-school children remains poorly understood. This paper investigates the epidemiology of parasitic infections in 460 pre-school children who were part of a larger case-control study of severe malaria in Kilifi on the Kenyan coast. Almost one-third (28.7%) were infected with hookworm, 20.2% with Ascaris lumbricoides and 15.0% with Trichuris trichiura. Infection prevalence of each species rose with age, and the prevalence of heavy infection with hookworm and mean intensity of hookworm were markedly age-dependent. One-third (34.3%) of children had malaria. Overall, 76.3% of children were anaemic (haemoglobin < 110 g/L), with the prevalence decreasing with age. Anaemia was significantly worst in children with heavy hookworm infection (> 200 eggs per gram). This relationship held for all ages, both sexes, and was independent of socioeconomic factors. The application of attributable morbidity methods confirmed the contribution of hookworm infection to anaemia.

A polymorphism that affects OCT-1 binding to the TNF promoter region is associated with severe malaria.

Genetic variation in cytokine promoter regions is postulated to influence susceptibility to infection, but the molecular mechanisms by which such polymorphisms might affect gene regulation are unknown. Through systematic DNA footprinting of the TNF (encoding tumour necrosis factor, TNF) promoter region, we have identified a single nucleotide polymorphism (SNP) that causes the helix-turn-helix transcription factor OCT-1 to bind to a novel region of complex protein-DNA interactions and alters gene expression in human monocytes. The OCT-1-binding genotype, found in approximately 5% of Africans, is associated with fourfold increased susceptibility to cerebral malaria in large case-control studies of West African and East African populations, after correction for other known TNF polymorphisms and linked HLA alleles.

Intermittent sulphadoxine-pyrimethamine to prevent severe anaemia secondary to malaria in pregnancy: a randomised placebo-controlled trial.

BACKGROUND: In areas of endemic transmission, malaria in pregnancy is associated with severe maternal anaemia and low-birthweight babies. We studied the efficacy of intermittent treatment doses of sulphadoxine-pyrimethamine in preventing malaria and severe anaemia in pregnancy in a double-blind placebo-controlled trial among primigravid women living in Kilifi District, Kenya. METHODS: Between January, 1996, and April, 1997, 1264 primigravid women were recruited when they attended for antenatal care, and randomly assigned sulphadoxine-pyrimethamine (640) or placebo (624). Women received one, two, or three doses of study medication depending on the duration of gestation at enrolment. Primary outcome measures were severe anaemia (haemoglobin <8 g/dL) and malaria parasitaemia, assessed at 34 weeks of gestation. Analyses were based on intention to treat among women who had study blood tests at 34 weeks. FINDINGS: 30 (5.3%) of 567 women in the sulphadoxine-pyrimethamine group and 199 (35.3%) of 564 in the placebo group had peripheral parasitaemia (protective efficacy 85% [95% CI 78-90], p<0.0001). 82 (14.5%) and 134 (23.7%) had severe anaemia (protective efficacy 39% [22-52], p<0.0001). Even women who booked late and received only one dose of sulphadoxine-pyrimethamine benefited significantly from the intervention. The effects were seen both in women who owned insecticide-treated bednets and in women who did not. INTERPRETATION: Intermittent presumptive treatment with sulphadoxine-pyrimethamine is an effective, practicable strategy to decrease the risk of severe anaemia in primigravidae living in malarious areas.

Cognitive sequelae of severe malaria with impaired consciousness.

Although cerebral malaria is the most common acute encephalopathy arising in children in Africa little is known of its effect upon the longer-term cognitive development of survivors. In Kenya, we compared the performance of 87 survivors of severe malaria with impaired consciousness to matched community controls on a wide range of tasks, not less than 42 months post illness episode. The presence of cognitive impairment was then related to both the pattern of symptoms at the time of the acute illness and the presence of gross neurological impairment on discharge. Significant group differences were found in areas of cognitive functioning suggestive of widespread impairment in the development of the ability to initiate, plan and carry out tasks (the executive functions). On tasks of more discrete cognitive skills (information processing) there were no significant group differences, although impaired performance was found more frequently in the severe malaria group. The odds ratio associated with the development of cognitive impairment following severe malaria with impaired consciousness was found to be 4.48 (95% CI 1.22, 16.47). A combination of 4 signs (coma, hypoglycaemia, seizures, and absence of hyperpyrexia) proved to have greater accuracy than the presence of gross neurological sequelae in predicting cognitive impairment (95% vs 93% specificity, 67% vs 58% sensitivity).

Environmental and entomological risk factors for the development of clinical malaria among children on the Kenyan coast.

Several malariometric studies have examined the impact on human-vector contact of house construction, demographics, bed net and insect repellent use. However, few studies have documented the significance of these proximate determinants on the risks of clinical disease. We undertook a matched case-control study of the risks of both mild clinical malaria and severe life-threatening malaria according to a range of putative factors which would influence the frequency of child-vector encounters in Kilifi district on the Kenyan coast. Among 394 severe disease cases, 380 age-matched mild disease cases, and their respective location and age-matched community controls, we were unable to demonstrate any statistically significant effect upon disease outcome of house construction, presence of domestic animals, or bed net use. Higher population density within a 250 m radius of the homes conferred significant protection from the risks of developing severe malaria compared to community controls. The risks of developing severe malaria compared to the community controls and the transition from mild to severe disease were statistically significantly lower in those who reported use of mosquito coils, local repellents or aerosol insecticides. We concluded that it is likely that the impact of household features on disease outcome is dependent upon both the density of infecting mosquitoes and acquired immunity within a given locality.

A community randomized controlled trial of insecticide-treated bednets for the prevention of malaria and anaemia among primigravid women on the Kenyan coast.

The effectiveness of insecticide-treated bednets (ITBN) in preventing malaria and anaemia among primigravidae living in Kilifi District, Kenya, was assessed by a randomized controlled trial between September 1994 and November 1995. All residents within 28 community clusters received ITBN in July 1993, whilst residents of another 28 clusters served as contemporaneous controls. All resident primigravid women with singleton pregnancies attending antenatal care at Kilifi District Hospital were eligible for recruitment. 503 primigravidae were recruited. 91.4% were anaemic antenatally (Hb < 11 g/dl): 91.0% from the intervention arm and 92.0% from the control arm. Severe anaemia (Hb < 7 g/dl) was found among 15.1% of intervention women and 20.1% of control women (P = 0.28). No significant differences were observed in reports of febrile illness or the presence of chloroquine in the serum or peripheral parasitaemia during the third trimester between the two groups. In the women delivering in hospital (n = 130), there was no association between placental malaria infection and the intervention: 77.4% of placentas from control women had evidence of past or active infection, compared with 72.0% of placentas from intervention women (P = 0.76). Similarly, in the women delivering in hospital, ITBN did not improve birth weight, and there were no differences in perinatal mortality between the two study groups. Despite ITBN having a great impact on paediatric severe malaria and mortality in this transmission setting, there was very little impact of ITBN on the morbidity associated with malaria infection in primigravidae. Alternative strategies are required to tackle this continued public health problem for pregnant women living in endemic areas similar to the Kenyan Coast.