点击显示 收起
Kenya Medical Research Institute/Wellcome Trust Programme, Centre for Geographic Medicine Research, Coast, Kilifi District Hospital, Kilifi, Kenya
Nuffield Department of Medicine, Department of Paediatrics
Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford
MRC Tropical Epidemiology Group, London School of Hygiene and Tropical Medicine, London, United Kingdom
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 ) 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.
Malaria causes >200 million episodes of febrile illness and >1 million deaths every year in young children living in sub-Saharan Africa [1, 2]. The factors determining which children die and which survive are complex, but they are likely related to both the host and the parasite. Of the host-specific factors, the sickle cell trait (HbAS) remains the best described [3], having been shown to confer strong protection against Plasmodium falciparum malaria in numerous studies conducted in various countries over the course of >50 years [410]; nevertheless, the protective mechanisms at work remain incompletely understood. A number have been proposed, including reduced parasite growth [11, 12] and enhanced removal of parasitized cells through innate [13, 14] or acquired [1518] immune processes; however, which are relevant in vivo and whether P. falciparum is the only selective agent remain unknown.
Of the many epidemiological studies conducted to date, most have had shortcomings; the majority have been small or cross-sectional and have provided only limited data on the prevalence of P. falciparum [19]. Case-control studies have focused on malaria and have provided few data on other diseases [47]. In addition, of the few cohort studies that have been conducted, none have provided data on falciparum malaria over its full range of clinical manifestations, and few have provided data on nonmalarial diseases [7, 15, 16, 20]. Therefore, with a view to filling in some of these gaps, we conducted 2 large cohort studies involving a total of >3000 children. These studies show the absence of any significant effect of HbAS on a wide range of childhood diseases and illustrate the specificity of protection against falciparum malaria.
PARTICIPANTS, MATERIALS, AND METHODS
Study design.
As outlined below, we conducted 2 studies that investigated the effect of HbAS on falciparum malaria and other childhood diseases, on the basis of clinical observations made in 2 cohorts of children living on the coast of Kenya.
Mild-disease cohort study.
The design of this study has been described in detail elsewhere [21, 22]. In brief, in September 1998, a cohort of 800 children and adults was recruited in the Ngerenya area of Kilifi District. This cohort was followed for clinical events by weekly active surveillance until August 2001. In addition, we tracked intercurrent clinical events at a dedicated research outpatient clinic. Children born into study households were recruited at birth. Participants exited the study if informed consent was withdrawn, if they moved out of the study area for >2 months, or if they died. During this interval, we conducted 4 cross-sectional surveys to assess the prevalence of P. falciparum. For the purpose of the present analysis, children were censored on their fifth birthday; this analysis includes 323 children who were <5 years old for >1 week during the study period.
Birth cohort study.
This study, which formed the basis of a randomized, controlled community trial of insecticide-impregnated bed nets, was conducted as described elsewhere [2325]. In brief, all children born within a defined rural study area (to the north of Kilifi District Hospital ) between May 1992 and April 1995 were recruited into a fixed birth cohort through a system of demographic surveillance involving visits every 6 weeks, by which all births, deaths, and migration events were recorded. Between May 1992 and December 1997, members of this cohort were identified on admission to the pediatric ward at KDH, the hospital facility closest to the study area. Routine collection of blood samples was not part of the original study; however, between May and October 2000, 2695 resident surviving members of the cohort were identified and were invited to provide a blood sample for hemoglobin genotyping. Genotyping was successfully completed for 2655 children.
Clinical definitions.
For both studies, at the time of presentation, a trained clinician who had access to appropriate clinical and laboratory tests obtained a detailed clinical history, conducted an examination, and recorded the results. Comorbid presentations were common in both studies, and the clinician could record up to 3 diagnoses for each child; however, for the purpose of the present analysis, we considered only the primary diagnoses leading to consultation or admission. We defined malaria in a number of ways. For the mild-disease cohort study, we defined symptomless parasitemia as a slide positive for P. falciparum in the absence of fever or other symptoms of clinical illness. For mild clinical malaria, we used 3 different definitions. Definition 1 was based on the diagnosis made by the trained clinician without the constraint of specific clinical or laboratory features; definition 2 was based on fever (axillary temperature of >37.50°C) in conjunction with a slide positive for blood-stage asexual P. falciparum at any parasite density (regardless of the age of the child); and definition 3 was based on fever in conjunction with a slide positive for blood-stage asexual P. falciparum at any parasite density for children <1 year old and at a parasite density of >2500 parasites/L for children 1 year old. Definition 3 was derived by multiple logistic regression methods, as described elsewhere [26], and accords with both a sensitivity and a specificity for clinical malaria of >85% in both subjects with HbAA and those with HbAS (T.W.M., unpublished data). On admission to the hospital, malaria was considered to be the primary diagnosis if P. falciparum was found in the peripheral blood and if other likely causes of clinical presentation could be excluded. The following definitions (which are modifications of those described elsewhere [27]) were used for severe malaria: (1) comathe inability to localize a painful stimulus, assessed >1 h after a seizure or the administration of anticonvulsants and after correction of hypoglycemia; (2) prostrationthe inability to breast-feed or sit without assistance; (3) multiple seizures2 or more seizures within 24 h of admission; (4) severe malarial anemiahemoglobin level of <5.0 g/dL in association with a parasite density of >10,000 parasites/L; and (5) hyperparasitemiaan episode of malaria in which >20% of red blood cells are infected with P. falciparum. Upper respiratory tract infection was diagnosed in children whose principal symptoms were characterized by choryza or pharyngitis and who had no other features of malaria. Acute lower respiratory tract infection was diagnosed in children who fulfilled the World Health Organization clinical criteria for pneumonia [28] if tests and the subsequent clinical course of disease supported this diagnosis. Gastroenteritis was defined as diarrhea (3 or more watery stools/day) with or without vomiting (3 or more episodes/day). In the mild-disease cohort study, slide-negative fever was defined as an axillary temperature of >37.50°C in a child whose slide was negative for P. falciparum and who had not received treatment with an antimalarial drug within the preceding 21 days; this definition took no account of the primary or secondary diagnosis and, therefore, encapsulated febrile episodes of a range of nonmalarial causes. Fever of unknown cause was a diagnosis of exclusion and was allocated to children whose slide was negative for P. falciparum and whose fever had no obvious etiology. Helminth infection was diagnosed in children who had a history of passing worms of any species, and skin infection was diagnosed in children who presented with a range of dermatological conditions, including scabies, boils, and impetigo. In the birth cohort study, malnutrition was diagnosed in children whose weight-for-height or weight-for-age was <70% or <60%, respectively, of the US National Center for Health Statistics reference medians or who had signs of marasmus or kwashiorkor. Nonmalarial anemia was diagnosed in children who presented with a hemoglobin level of <5.0 g/dL and without evidence of malarial infection.
Laboratory procedures.
Blood films were stained and examined for P. falciparum by standard methods. Parasite densities were recorded as a ratio of parasites to white blood cells or, for heavier infections, to red blood cells. Densities (number of parasites per microliter of whole blood) were calculated with data from full hematological assessments, if available, or, if not, on the assumption of a white blood cell count of 8 × 103 cells/L or a red blood cell count of 5 × 106 cells/L. Hemoglobin genotypes were characterized by electrophoresis.
Statistical analysis.
Odds ratios (ORs) for the prevalence of symptomless parasitemia in children with HbAS versus children with HbAA were derived by both univariate and multivariate logistic regression analysis. Multivariate analyses included age and season (defined in 3-month blocks). We accounted for the potential clustering of symptomless-parasitemia events within individual study children by using the sandwich estimator (as described by Armitage et al. [29]), which inflates confidence intervals (CIs) and adjusts P values as appropriate. Log-transformed parasite densities in P. falciparumpositive children with HbAA and those with HbAS were compared within each clinical classification by linear regression analysis, with adjustment for within-participant clustering of events and both with and without adjustment for the confounding variables age, proximity to the nearest health center, and season. We derived incidence rate ratios (IRRs) for malaria and other diseases in children with HbAS versus those with HbAA within each cohort separately, using Poisson regression. For both cohort studies, our final models included the following explanatory variables: hemoglobin genotype, age (as a continuous variable), sex, season, and ethnic group. For the birth cohort study, we also adjusted for bed-net usage (by randomization arm), proximity to the nearest health center, and access to the hospital by bus. We used the likelihood ratio test to assess interactions between explanatory variables, as appropriate; no significant interactions were found. For the purpose of the present analysis, children in the mild-disease cohort were considered not to be at risk for malaria (and were omitted from both the numerator and denominator populations) for 21 days after receiving treatment with an antimalarial drug. All children were censored on their fifth birthday. In all of our analyses, we controlled for the duration of follow-up of study children. All analyses were conducted by use of STATA (version 8.0; Timberlake).
Permission with respect to ethics was granted for both studies by the Kenya Medical Research Institute National Ethical Review Committee. Written, informed consent was provided by all study participants or their parents.
RESULTS
In the mild-disease cohort study, 4254 clinic visits were made by 323 children during 451.9 child-years of follow-up (cyfu). The following 7 diagnoses accounted for >90% of the consultations: mild clinical malaria (definition 1) (1288/4254 [30%]), upper respiratory tract infection (1168/4254 [27%]), skin infection (571/4254 [13%]), lower respiratory tract infection (539/4254 [13%]), gastroenteritis (478/4254 [11%]), fever of unknown cause (244/4254 [6%]), and helminth infection (170/4254 [4%]). The numbers of children who received other diagnoses were too few to permit meaningful comparisons. Only 1 child died, a participant with HbAA who had been suffering from malnutrition. In the birth cohort study, a total of 1145 hospital admissions were recorded among 2655 children during 10,381 cyfu. Malaria (561/1145 [49%]), lower respiratory tract infection (310/1145 [27%]), and gastroenteritis (91/1145 [8%]) accounted for 84% of all admissions. Of the 561 children with malaria, 197 (35%) showed 1 or more signs of severity. Of the 197 episodes, 182 (92%) could be defined according to 1 of 3 categories: cerebral malaria, severe malarial anemia with >10,000 parasites/L, and malaria with convulsions (2 or more seizures during the previous 24 h). The numbers of children admitted with other diagnoses or malarial syndromes were too few to permit meaningful comparisons. Because the hemoglobin genotyping for this cohort was conducted at the end of the study, it was not possible to investigate the contribution of genotype to death. No children with sickle cell disease (HbSS) were detected in the mild-disease cohort, but 3 children with HbSS were detected in the birth cohort. None of these children were admitted to the hospital during the study period.
HbAS and the risk of malaria.
We found no evidence for any effect of HbAS on the prevalence of symptomless parasitemia: the prevalence during the 4 cross-sectional surveys combined was 95 (14.8%) of 643 in children with HbAA and 13 (14.4%) of 90 in children with HbAS (adjusted OR, 0.96 [95% CI, 0.442.07]); P = .91). However, both the incidence of mild clinical malaria in the community and of hospital admission for severe malaria were significantly lower in children with HbAS than in children with HbAA, the degree of protection being roughly twice as great for the latter than the former (tables 1 and 2). HbAS was associated with a similar degree of protection against mild clinical malaria for each of the 3 definitions considered (table 1). Conversely, in the case of hospital admissions for malaria, HbAS was in general associated with increasing protection against episodes of increasing severity: although the incidence of all admissions for malaria was reduced by 75%, the incidence of admissions for cerebral malaria was reduced by 86%, and the incidence of admissions for severe malarial anemia was reduced by almost 90% (table 2).
Parasite densities during incident events.
We found no significant effect of HbAS on geometric mean densities of P. falciparum parasites during episodes of symptomless parasitemia, either before or after adjustment for the effects of age, proximity to the nearest health center, and season (ratio, 1.27 [95% CI, 0.285.89]; P = .747). However, parasite densities were significantly lower during episodes of both mild clinical malaria (ratio, 0.49 [95% CI, 0.290.83]; P = .009) and severe malaria that required a child to be admitted to the hospital (ratio, 0.23 [95% CI, 0.100.52]; P < .0001) (figure 1).
Nonmalarial diseases.
We found no significant associations between HbAS and the incidence of any other diseases, whether detected at the outpatient clinic or on hospital admission (tables 1 and 2). Of particular note, in the mild-disease cohort study, we saw no effect of HbAS on the incidence of documented febrile episodes (of any cause) in the absence of malaria (IRR, 1.02 [95% CI, 0.841.26]; P = .78) or on the incidence of upper or lower respiratory tract infections. The low IRRs for admission to the hospital for gastroenteritis (IRR, 0.59) and severe anemia without malarial parasites (IRR, 0.35) were based on very few observations, and neither reached statistical significance (table 2).
DISCUSSION
The protective effect of HbAS against falciparum malaria has been the subject of speculation and debate for >50 years. After early scepticism [30, 31, 32], it is now widely accepted that the most likely explanation for the current frequencies of HbAS in many tropical populations is selection by P. falciparum; however, the epidemiological evidence supporting this conclusion remains incomplete. For example, although the available data suggest that HbAS confers greater protection against severe forms of malaria than against mild forms, these data are difficult to interpret, because they originate from many studies that investigated limited aspects of falciparum malaria and that were conducted in areas of varying transmission (reviewed in Roberts et al. [19]). Moreover, very few studies have investigated the degree to which the protection conferred by HbAS is specific to malaria. These issues are important, not only because they may be informative with respect to the mechanisms by which HbAS confers protection against malaria, but because they may help us to better understand the relationships between HbAS, malaria, and other childhood diseases. It was for these reasons that we conducted the 2 large cohort studies described in this article.
In agreement with the findings of most other studies, we found no evidence that HbAS protects against symptomless parasitemia, in terms of either prevalence or parasite density. However, HbAS was 50% protective against mild clinical malaria, and parasite densities during such episodes were significantly lower in children with HbAS than in those with HbAA. Protection reached 75% against hospital admission for falciparum malaria and almost 90% against severe malarial episodes. Two observations were particularly striking: first, of the children admitted to the hospital for falciparum malaria, those with HbAS had parasite densities >4-fold lower than those with HbAA; and second, the protective effect of HbAS was equally strong against the 2 most common forms of severe malariacerebral malaria and severe malarial anemia. These observations support the concept that HbAS confers protection against severe malaria by limiting disease progression: infections less often progress to the point at which either symptoms are evident (mild disease) or complications ensue (severe disease). This could equally be mediated by the reduced ability of parasites to grow and multiply in HbAS cells [11, 12] or by their early removal from the circulation. A number of mechanisms have been proposed for the latter. First, parasite-infected HbAS erythrocytes have been shown to sickle 6 times more readily than nonparasitized HbAS cells [33, 34], a phenomenon that may lead to intracellular parasite death [35] and/or their enhanced removal by the immune system. Although the latter may be largely the result of innate immunity [14, 36, 37], recent data have suggested that acquired immunity may also be involved [1518]. Which of these processes are relevant in vivo and what their relative contributions to conferring protection against malaria might be remain to be determined.
The lack of any measurable effect of HbAS on most other childhood diseases observed in the present analysis is interesting. Although HbAS has no appreciable impact on health in developed countries [38], we expected to find some impact on other childhood diseases in our cohorts, for a number of reasons. First, in many tropical settings, it can be difficult to differentiate among the common causes of childhood diseases on the basis of clinical criteria alone. For example, respiratory distress is a common feature in children with pneumonia, malaria, and other severe illnesses, and, therefore, differentiating among such diseases can be difficult in areas in which a large proportion of children are parasitemic [39, 40]. Even if the effect of HbAS were absolutely specific to malaria, we would still have expected a less clear-cut result simply because of some degree of misclassification. Second, we thought that HbAS might result in wider health benefits via a reduction in the sequelae of malarial infections. For example, malaria is a recognized cause of malnutrition [22, 4143], which in turn is a major determinant of disease susceptibility [44, 45]. Similarly, malaria might enhance the severity of other comorbid infectious diseases via direct effects, such as immune suppression, or via enhancement of bacterial invasion [46]. Indeed, in a number of well-documented cases, effective malaria-control programs have lead to reductions in mortality several fold higher than what was expected on the basis of prior estimates of malaria-specific mortality [4751], and estimates of the differential mortality required to explain the current prevalence of the HbS gene are also significantly greater than those attributed to malaria alone [46]. However, in both of these cases, it is impossible to determine the relative contributions of factors such as misclassification, potentiation of comorbid events, the nonspecificity of malaria control interventions, and genetic effects.
Despite these considerations, we found that the protection conferred by HbAS was strikingly specific to falciparum malaria. If endemic malaria really has a nonspecific impact on all-cause mortality, why was this not reflected by reductions in 1 or more categories of common nonmalarial events Two explanations seem plausible. The first explanation is that the pathophysiological processes that result in death are largely silent and do not give rise to well-circumscribed clinical presentations. Two processes seem most likely in this regard: anemia and invasive bacterial disease.
Anemia is a common sequela of malarial infections but is often clinically silent [52, 53]. As such, it likely makes a large but hidden contribution to overall mortality due to malaria, especially in young children [52]. The pathogenesis of malaria-related anemia is multifactorial, involving both bone-marrow suppression and acute hemolysis [52, 54]. Mortality is greatest when anemia is severe (<5 g/dL hemoglobin) and is complicated by other signs of severity [55]. Two factors correlate best with the development of severe anemia: hemoglobin level preceding the malaria transmission season and the parasite density achieved during incident infection [56]. It seems likely that children with HbAS enjoy a double advantage in this regard: first, because they suffer fewer clinical attacks of malaria, their baseline hemoglobin levels may be higher; and second, they may be further protected by the lower parasite densities achieved during incident infection. This conclusion is supported by the protection conferred against severe anemia seen in both the present analysis and in previous studies [7]. Similarly, although it is probable that a high proportion of the deaths that occur during childhood involve invasive bacterial infections, these often present nonspecifically and can be rapidly fatal; therefore, they are difficult to quantify in facility-based studies [57]. Blood cultures were not done as part of our routine assessment of children during our 2 studies; as a result, we might well have failed to detect an effect of HbAS on bacteremia, even if one existed.
A second explanation for failing to detect a true effect of HbAS on nonmalarial disease relates to its lack of protection against symptomless parasitemia. It seems possible that symptomless malarial parasitemia, as opposed to symptomatic malarial infection, is the main predisposing factor for death due to other diseases. In areas of stable transmission, the bulk of the malarial disease pyramid lies in symptomless parasitemia in children <5 years old. Under these circumstances, it is hard to judge the relative effects of silent and symptomatic events on the risk of comorbid diseaseeven a small effect of symptomless parasitemia on the risk of other diseases in an individual could have a much greater effect at the population level, given the relative prevalence of these events. Under these circumstances, HbAS might not give rise to a visible effect on nonmalarial diseases, because it does not protect against symptomless parasitemia.
It can be seen from the above discussion that observational studies of the kind reported here can generate more questions than answers; nevertheless, we still believe that exploration of the relationships between genetic traits, malaria, and other morbid events is useful. In light of the results of the present analysis and other studies, it seems likely that the protection conferred by HbAS is remarkably specific to malaria. This does not appear to be true for + thalassemia, in which homozygotes are equally protected against malaria and other infectious diseases [58]. Following some of nature's clues may yet yield rewards in terms of developing a better understanding the pathophysiological processes of malaria and their interactions with the processes of other diseases.
Acknowledgments
We thank Chris Newbold, Norbert Peshu, Brett Lowe, David Weatherall, and David Roberts, for helpful advice; Amanda Ross, for statistical support; and the clinical and medical officers, nurses, and field and laboratory staff of the Kenya Medical Research Institute, Centre for Geographic Medicine Research, Coast, for their help with data collection. This article is dedicated to Prof. Steve Bennett, a key collaborator who died in April 2003, before completion of the analysis of the studies.
References
1. Murray CJ, Lopez AD. Global mortality, disability, and the contribution of risk factors: Global Burden of Disease Study. Lancet 1997; 349:143642. First citation in article
2. Snow RW, Craig MH, Newton CRJC, Steketee RW. The public health burden of P. falciparum malaria in Africa: deriving the numbers. Bethesda, MD: Fogerty International Center, National Institutes of Health, 2003. First citation in article
3. Allison AC. Polymorphism and natural selection in human populations. Cold Spring Harb Symp Quant Biol 1964; 29:13749. First citation in article
4. Gilles HM, Fletcher KA, Hendrickse RG, Lindner R, Reddy S, Allan N. Glucose-6-phosphate-dehydrogenase deficiency, sickling, and malaria in African children in South Western Nigeria. Lancet 1967; 1:13840. First citation in article
5. Willcox M, Bjorkman A, Brohult J, Pehrson PO, Rombo L, Bengtsson E. A case-control study in northern Liberia of Plasmodium falciparum malaria in haemoglobin S and beta-thalassaemia traits. Ann Trop Med Parasitol 1983; 77:23946. First citation in article
6. Hill AV, Allsopp CE, Kwiatkowski D, et al. Common west African HLA antigens are associated with protection from severe malaria. Nature 1991; 352:595600. First citation in article
7. Aidoo M, Terlouw DJ, Kolczak MS, et al. Protective effects of the sickle cell gene against malaria morbidity and mortality. Lancet 2002; 359:13112. First citation in article
8. Beet EA. Sickle cell disease in the Balovale district of northern Rhodesia. East Afr Med J 1946; 23:7586. First citation in article
9. Beet EA. Sickle cell disease in northern Rhodesia. East Afr Med J 1947; 24:21222. First citation in article
10. Allison AC. Protection afforded by sickle cell trait against subtertian malarial infection. Br Med J 1954; 1:2905. First citation in article
11. Friedman MJ. Erythrocytic mechanism of sickle cell resistance to malaria. Proc Natl Acad Sci USA 1978; 75:19947. First citation in article
12. Pasvol G, Weatherall DJ, Wilson RJ. Cellular mechanism for the protective effect of haemoglobin S against P. falciparum malaria. Nature 1978; 274:7013. First citation in article
13. Luzzatto L, Pinching AJ. Commentry to R. L. Nagel: innate resistance to malaria: the intraerythrocytic cycle. Blood Cells 1990; 16:3407. First citation in article
14. Ayi K, Turrini F, Piga A, Arese P. Enhanced phagocytosis of ring-parasitized mutant erythrocytes: a common mechanism that may explain protection against falciparum-malaria in sickle-trait and beta-thalassemia-trait. Blood 2004; 104:336471. First citation in article
15. Marsh K, Otoo L, Hayes RJ, Carson DC, Greenwood BM. Antibodies to blood stage antigens of Plasmodium falciparum in rural Gambians and their relation to protection against infection. Trans R Soc Trop Med Hyg 1989; 83:293303. First citation in article
16. Le Hesran JY, Personne I, Personne P, et al. Longitudinal study of Plasmodium falciparum infection and immune responses in infants with or without the sickle cell trait. Int J Epidemiol 1999; 28:7938. First citation in article
17. Abu-Zeid YA, Abdulhadi NH, Theander TG, et al. Seasonal changes in cell mediated immune responses to soluble Plasmodium falciparum antigens in children with haemoglobin AA and haemoglobin AS. Trans R Soc Trop Med Hyg 1992; 86:202. First citation in article
18. Bayoumi RA, Abu-Zeid YA, Abdulhadi NH, et al. Cell-mediated immune responses to Plasmodium falciparum purified soluble antigens in sickle-cell trait subjects. Immunol Lett 1990; 25:2439. First citation in article
19. Roberts DJ, Harris T, Williams T. The influence of inherited traits on malaria infection. In: Bellamy R, ed. Susceptibility to infectious diseases: the importance of host genetics. Cambridge: Cambridge University Press, 2004:13984. First citation in article
20. Fleming AF, Storey J, Molineaux L, Iroko EA, Attai ED. Abnormal haemoglobins in the Sudan savanna of Nigeria. I. Prevalence of haemoglobins and relationships between sickle cell trait, malaria and survival. Ann Trop Med Parasitol 1979; 73:16172. First citation in article
21. Mwangi TW. Clinical epidemiology of malaria under differing levels of transmission. Oxford: Open University, 2003:326. First citation in article
22. Nyakeriga AM, Troye-Blomberg M, Chemtai AK, Marsh K, Williams TN. Malaria and nutritional status in children living on the coast of Kenya. Am J Clin Nutr 2004; 80:160410. First citation in article
23. Snow RW, Howard SC, Mung'Ala-Odera V, et al. Paediatric survival and re-admission risks following hospitalization on the Kenyan coast. Trop Med Int Health 2000; 5:37783. First citation in article
24. Nevill CG, Some ES, Mung'ala VO, et al. Incecticide-treated bednets reduce mortality and severe morbidity from malaria among children on the Kenyan coast. Trop Med Int Health 1996; 1:13946. First citation in article
25. Snow RW, McCabe E, Mbogo CN, et al. The effect of delivery mechanisms on the uptake of bed net re-impregnation in Kilifi District, Kenya. Health Policy Plan 1999; 14:1825. First citation in article
26. Smith T, Schellenberg JA, Hayes R. Attributable fraction estimates and case definitions for malaria in endemic areas. Stat Med 1994; 13:234558. First citation in article
27. World Health Organization, Division of Control and Tropical Diseases. Severe and complicated malaria, 2nd ed. Trans R Soc Trop Med Hyg 1990; 84(Suppl 2):S165. First citation in article
28. World Health Organization. Acute respiratory infections in children: case management in small hospitals in developing countries: a manual for doctors and other senior health workers [WHO/ARI 90.5]. Geneva: WHO, 1990. First citation in article
29. Armitage P, Berry G, Matthews JNS. Using STATA's robust cluster command as appropriate: statistical methods in medical research. 4th ed. Oxford: Blackwell Scientific Publications, 2001. First citation in article
30. Beutler E, Dern RJ, Flanagan CL. Effect of sickle-cell trait on resistance to malaria. Br Med J 1955; 1:118991. First citation in article
31. Foy H, Kondi A, Timms GL, Brass W, Bushra F. The variability of sickle-cell rates in the tribes of Kenya and the Southern Sudan. Br Med J 1954; 1:2947. First citation in article
32. Walters JH, Bruce-Chwatt LJ. Sickle-cell anaemia and falciparum malaria. Trans R Soc Trop Med Hyg 1956; 50:5114. First citation in article
33. Luzzatto L, Nwachuku-Jarrett ES, Reddy S. Increased sickling of parasitised erythrocytes as mechanism of resistance against malaria in the sickle-cell trait. Lancet 1970; 1:31921. First citation in article
34. Roth EF Jr, Friedman M, Ueda Y, Tellez I, Trager W, Nagel RL. Sickling rates of human AS red cells infected in vitro with Plasmodium falciparum malaria. Science 1978; 202:6502. First citation in article
35. Friedman MJ, Roth EF, Nagel RL, Trager W. Plasmodium falciparum: physiological interactions with the human sickle cell. Exp Parasitol 1979; 47:7380. First citation in article
36. Shear HL, Roth EF Jr, Fabry ME, et al. Transgenic mice expressing human sickle hemoglobin are partially resistant to rodent malaria. Blood 1993; 81:2226. First citation in article
37. Hebbel RP. Sickle hemoglobin instability: a mechanism for malaria protection. Redox Report 2003; 8:23840. First citation in article
38. Beutler E. The sickle cell diseases and related disorders. In: Beutler E, ed. Williams hematology. 6th ed. New York: McGraw-Hill, 2001:5945. First citation in article
39. Mulholland K. Magnitude of the problem of childhood pneumonia. Lancet 1999; 354:5902. First citation in article
40. Williams BG, Gouws E, Boschi-Pinto C, Bryce J, Dye C. Estimates of world-wide distribution of child deaths from acute respiratory infections. Lancet Infect Dis 2002; 2:2532. First citation in article
41. McGregor IA. Malaria: nutritional implications. Rev Infect Dis 1982; 4:798804. First citation in article
42. McGregor IA, Gilles HM, Walters JH, Davies AH, Pearson FA. Effects of heavy and repleated malarial infections on Gambian infants and children: effects of erythrocyte parasitization. Br Med J 1956; 32:68692. First citation in article
43. Friedman JF, Phillips-Howard PA, Hawley W, et al. Impact of permethrin-treated bed nets on growth, nutritional status, and body composition of primary school children in western Kenya. Am J Trop Med Hyg 2003; 68:7885. First citation in article
44. Ezzati M, Lopez AD, Rodgers A, Vander Hoorn S, Murray CJ. Selected major risk factors and global and regional burden of disease. Lancet 2002; 360:134760. First citation in article
45. Pelletier DL, Frongillo EA Jr, Schroeder DG, Habicht JP. The effects of malnutrition on child mortality in developing countries. Bull World Health Organ 1995; 73:4438. First citation in article
46. Molineaux L. Malaria and mortality: some epidemiological considerations. Ann Trop Med Parasitol 1997; 91:81125. First citation in article
47. Molineaux L. The impact of parasitic diseases and their control, with an emphasis on malaria and Africa. In: Vallin J, Lopez AD, eds. Health policy and mortality prospects. Liège, Belgium: Ordina Editions, 1985:1344. First citation in article
48. Greenwood BM, Bradley AK, Greenwood AM, et al. Mortality and morbidity from malaria among children in a rural area of The Gambia, West Africa. Trans R Soc Trop Med Hyg 1987; 81:47886. First citation in article
49. Greenwood BM, Greenwood AM, Bradley AK, et al. Comparison of two strategies for control of malaria within a primary health care programme in The Gambia. Lancet 1988; 1:11217. First citation in article
50. Alonso PL, Lindsay SW, Armstrong Schellenberg JR, et al. A malaria control trial using insecticide-treated bed nets and targeted chemoprophylaxis in a rural area of The Gambia, west Africa. VI. The impact of the interventions on mortality and morbidity from malaria. Trans R Soc Trop Med Hyg 1993; 87(Suppl 2):S3744. First citation in article
51. Giglioli G. Changes in the pattern of mortality following the eradication of hyperendemic malaria from a highly susceptible community. Bull World Health Organ 1972; 46:181202. First citation in article
52. Menendez C, Fleming AF, Alonso PL. Malaria-related anaemia. Parasitol Today 2000; 16:46976. First citation in article
53. Menendez C, Kahigwa E, Hirt R, et al. Randomised placebo-controlled trial of iron supplementation and malaria chemoprophylaxis for prevention of severe anaemia and malaria in Tanzanian infants. Lancet 1997; 350:84450. First citation in article
54. Weatherall DJ, Abdalla S. The anaemia of Plasmodium falciparum malaria. Br Med Bull 1982; 38:14751. First citation in article
55. Marsh K, Forster D, Waruiru C, et al. Indicators of life-threatening malaria in African children. N Engl J Med 1995; 332:1399404. First citation in article
56. Dicko A, Klion AD, Thera MA, et al. The etiology of severe anemia in a village and a periurban area in Mali. Blood 2004; 104:1198200. First citation in article
57. Berkley JA, Lowe BS, Mwangi I, et al. Community acquired bacteremia amongst children admitted to a rural Kenyan district hospital. N Engl J Med 2005; 352:3947. First citation in article
58. Allen SJ, O'Donnell A, Alexander ND, et al. Alpha+thalassemia protects children against disease caused by other infections as well as malaria. Proc Natl Acad Sci USA 1997; 94:1473641. First citation in article