Both heterozygous and homozygous + thalassemias protect against severe and fatal Plasmodium falciparum malaria on the coast of Kenya
http://www.100md.com
《血液学杂志》
the Kenya Medical Research Institute (KeMRI), Centre for Geographic Medicine Research, Kilifi, Kenya
Nuffield Department of Clinical Medicine and Department of Paediatrics, University of Oxford, John Radcliffe Hospital
Neurosciences Unit, Institute of Child Health, University College, London
Department of Paediatrics, Faculty of Medicine and the Wellcome Trust Centre for Clinical Tropical Medicine, Imperial College, London, United Kingdom.
Abstract
Although the + thalassemias almost certainly confer protection against death from malaria, this has not been formally documented. We have conducted a study involving 655 case patients with rigorously defined severe malaria and 648 controls, frequency matched on area of residence and ethnic group. The prevalence of both heterozygous and homozygous + thalassemia was reduced in both case patients with severe malaria (adjusted odds ratios [ORs], 0.73 and 0.57; 95% confidence intervals [95% CIs], 0.57-0.94 and 0.40-0.81; P = .013 and P = .002, respectively, compared with controls) and among the subgroup of children who died after admission with severe malaria (OR, 0.60 and 0.37; 95% CI, 0.37-1.00 and 0.16-0.87; P = .05 and P = .02, respectively, compared with surviving case patients). The lowest ORs were seen for the forms of malaria associated with the highest mortality—coma and severe anemia complicated by deep, acidotic breathing. Our study supports the conclusion that both heterozygotes and homozygotes enjoy a selective advantage against death from Plasmodium falciparum malaria.
Introduction
The + thalassemias are the commonest known genetic disorder in humans,1 a situation that probably reflects a selective advantage against death from malaria. However, to date, the protection they afford against severe and fatal falciparum malaria has been investigated in only 2 studies, and these have yielded conflicting results.2,3 Here we describe a large case-control study in which we have found that both heterozygous and homozygous + thalassemia are associated with a reduced risk for severe and fatal Plasmodium falciparum malaria in children living on the coast of Kenya.
Study design
Case patients with severe malaria and controls
The study was conducted in Kilifi district, where the population is composed predominantly of rural dwellers of the Mijikenda ethnolinguistic group. The epidemiology and clinical characteristics of malaria in the study area have been described in detail previously.4,5 Case patients were children with clinical features of severe P falciparum malaria who were admitted to the high-dependency unit (HDU) at Kilifi District Hospital (KDH), Kenya, between January 2001 and August 2003. We use 3 clinical features to define severe malaria at our hospital: prostration, coma (Blantyre coma score = 2), and deep breathing. At least 1 of these features is present on admission in approximately 98% of our inpatients who die of malaria.6 Children were eligible for the study if they met the following criteria: (1) P falciparum malaria with any of the 3 clinical features of severity; (2) residence within the study area of the Kenya Medical Research Institute (KeMRI) Centre for Geographic Medicine Research, Coast, Kilifi, an area populated by more than 200 000 subjects that has been under continuous demographic surveillance since 20017; and (3) Mijikenda ethnic origin. Children with uncomplicated severe malaria anemia (SMA) (ie, those with P falciparum malaria, in association with a hemoglobin level lower than 5 g/dL but with no signs of respiratory distress) are not routinely treated in our HDU because the inpatient mortality rate in this group is less than 1%.6 As a result, such children were not included as case patients. All case patients were treated according to standard guidelines, as described in detail previously.8 Controls were residents of the same study area who were randomly selected on the basis of ethnic group (Mijikenda) and who were frequency matched to case patients on the basis of location. Controls were younger than 7, but no attempt was made to match on age. The KeMRI National ethical Review Committee approved the study. Individual written informed consent was provided by all study participants or their parents.
Laboratory methods
Routine hematologic and biochemical data are collected on all children admitted to the HDU at KDH, and malaria parasite densities are determined by standard methods, as described previously.8 Case patients and controls were typed for the common African 3.7-kb -globin deletion by polymerase chain reaction (PCR), as described previously,9 using DNA extracted by standard methods (Qiagen DNA Blood Mini Kit; Qiagen, West Sussex, United Kingdom; or Puregene; Gentra Systems, Minneapolis, MN).
Statistical analysis
Categorical data were analyzed using the 2 test for trend, and numerical data were compared using analysis of variance (ANOVA). We determined the odds ratios (ORs) for severe malaria, severe malaria subgroup, and inpatient death by + thalassemia genotype using logistic regression, with and without adjustment for Mijikenda ethnic subgroup, age, and sex.
Results and discussion
Our study included 655 case patients and 648 controls. The + thalassemia gene frequencies in case patients and controls and the presenting characteristics of case patients are summarized in Tables 1, 2, and 3. The prevalence of both heterozygous and homozygous + thalassemia was significantly lower in case patients than in controls, and the lowest ORs were seen for homozygosity in children with the syndromes associated with the highest risk for mortality in our population—symptomatic SMA10 and deep breathing with coma6 (Table 4). Although admission hemoglobin concentrations were similar between genotypic groups (Table 3), the trend toward increasing hemoglobin values across the groups is noteworthy, given that heterozygous and homozygous + thalassemia are associated with anemia at steady state.11 We found no effect of + thalassemia on admission parasite density.
Simultaneous with the showing of an association between homozygous and heterozygous + thalassemia and protection from severe P falciparum disease, we found evidence for a protective association against death from malaria. Among children with severe malaria, admitted to our HDU on the basis of strictly defined clinical criteria, the risk for death in hospital was 40% lower in heterozygotes and more than 60% lower in homozygotes than in healthy children (Table 2). Furthermore, among case patients with fatal disease, severe acidosis (base deficit greater than 15) at admission was less common in homozygotes (1 of 7; 14%) than in healthy persons (17 of 33; 51%) or heterozygotes (14 of 32; 44%), though this trend did not reach statistical significance (2 = 3.5; P = .19).
The genotypic pattern of protection seen in our study was similar to that seen in Papua, New Guinea, where protection was greatest for anemia and acidosis2; however, in the latter study, protection did not reach significance in heterozygotes, and the in-hospital mortality rate was too low (15 of 433; 3.5%) to detect a between-genotype difference.2 Our observations are not, however, in accord with those from Ghana,3 where protection was seen in heterozygotes (OR, 0.74; 95% confidence interval [95% CI], 0.56-0.98; P = .03), but not in homozygotes (OR not reported), and where no protection was seen against in-hospital death despite a mortality rate (11%3) comparable with that seen in our study. These discrepancies may well be attributable to statistical considerations and may be explicable on the basis of the greater power of the current study than previous studies to detect significant differences; however, they may also relate to differences in the design and conduct of these studies. All 3 adopted a case-control design, an approach that is justified on the grounds that severe P falciparum malaria is a relatively rare outcome, affecting, for example, only approximately 10% of 1- to 5-year-olds in the Kilifi study area. Although case-control studies are open to problems of bias and confounding, we believe for several reasons that it is unlikely our results have been affected by such problems. More than 95% of residents of the Kilifi study area are of Mijikenda ancestry. Although this Bantu population can be categorized into 3 main subgroups—Giriama, Chonyi, and Kauma—we have detected no significant differences in the - allele frequencies between these groups in extensive surveys of more than 4000 children (T.N.W., unpublished data, collected between October 2000 and November 2004). Moreover, we adjusted for these ethnic subgroups in our logistic regression analysis, an exercise that made no material difference to our results. Finally, our controls were frequency-matched on location, through our population database, and our analysis was further adjusted for age. Nevertheless, despite these considerations, we cannot exclude the possibility that unknown biases might have affected our study, which should ideally be replicated using an alternative design such as a family-based or a prospective cohort approach.
What then can such studies tell us about the mechanism by which + thalassemia affords protection against malaria? A number of hypotheses have been proposed, including reduced parasite growth and invasion,12 increased phagocytosis of infected red blood cells,12 altered surface antigen expression that potentially augments immunologic clearance,13,14 and decreased parasite rosetting15; nevertheless, for these to be relevant, they must be compatible with observations from epidemiologic studies conducted under conditions of natural malaria transmission. The fact that, in keeping with all previous observations made in vivo,2,3,16-19 we found no effect at the level of parasite density makes it unlikely that + thalassemia protects simply by limiting the growth or increasing the clearance of parasites. Instead, it seems more likely that it acts by attenuating the consequences of malaria infection.
Severe and fatal malaria results from a range of interrelated pathophysiologic processes that affect multiple organ systems.6 These processes are thought to include the local and systemic release of various cytokines, anemia, decreased red cell deformability and adhesion phenomena such as sequestration and rosetting,20-23 and the clinical features of severe malaria (such as coma, acidosis, and hypovolemic shock), likely reflect the balance of such processes within individual patients.24 That + thalassemia protects against a range of clinical manifestations of severe malaria suggests it does so through a mechanism that is central to many of these processes. In this regard, results from a recent study are particularly intriguing. In an extension of the previous study in Papua, New Guinea,2 Cockburn and colleagues,25 made 2 related observations: first, that a promoter polymorphism of red cell complement receptor 1 (CR1), an important receptor for rosetting, was significantly associated with protection from severe falciparum malaria; second, that + thalassemia was independently associated with the reduced expression of red cell CR1. These observations support the earlier work of Carlson,15 who suggested that a number of the hemoglobinopathies might protect against severe malaria by reducing the ability of red cells to form rosettes. Further work will be required to explore these potentially important observations.
Acknowledgements
We thank the study subjects and their families for their cooperation, and we thank the medical, nursing, and support staff who assisted with this study. We thank David Weatherall, Brett Lowe, Norbert Peshu, and David Roberts for advice and support. This paper is published with the permission of the Director of KeMRI.
Footnotes
Prepublished online as Blood First edition Paper, March 15, 2005; DOI 10.1182/blood-2005-01-0313.
Supported by grants (T.N.W., C.R.J.C.N., K.M.) and a program grant (K.M., C.R.J.C.N.) from the Wellcome Trust, UK.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
References
Weatherall DJ, Clegg JB. The thalassaemia syndromes. 3rd ed. Oxford, United Kingdom: Blackwell Scientific Publications; 2002.
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 U S A. 1997;94: 14736-14741.
Mockenhaupt FP, ehrhardt S, Gellert S, et al. (+)Thalassaemia protects from severe malaria in African children. Blood. 2004;104: 2003-2006.
Snow RW, Omumbo JA, Lowe B, et al. Relation between severe malaria morbidity in children and level of Plasmodium falciparum transmission in Africa. Lancet. 1997;349: 1650-1654.
Mbogo CN, Snow RW, Kabiru eW, et al. Low-level Plasmodium falciparum transmission and the incidence of severe malaria infections on the Kenyan coast. Am J Trop Med Hyg. 1993;49: 245-253.
Marsh K, Forster D, Waruiru C, et al. Indicators of life-threatening malaria in African children. N engl J Med. 1995;332: 1399-1404.
Berkley JA, Lowe BS, Mwangi I, et al. Bacteremia among children admitted to a rural hospital in Kenya. N engl J Med. 2005;352: 39-47.
Maitland K, Pamba A, Newton CR, Levin M. Response to volume resuscitation in children with severe malaria. Pediatr Crit Care Med. 2003;4: 426-431.
Chong SS, Boehm CD, Higgs DR, Cutting GR. Single-tube multiplex-PCR screen for common deletional determinants of alpha-thalassemia. Blood. 2000;95: 360-362.
english M, Waruiru C, Amukoye e, et al. Deep breathing in children with severe malaria: indicator of metabolic acidosis and poor outcome. Am J Trop Med Hyg. 1996;55: 521-524.
Williams TN, Maitland K, Ganczakowski M, et al. Red blood cell phenotypes in the alpha + thalassaemias from early childhood to maturity. Br J Haematol. 1996;95: 266-272.
Yuthavong Y, Butthep P, Bunyaratvej A, Fucharoen S, Khurmith S. Impaired parasite growth and increased susceptibility to phagocytosis of Plasmodium falciparum infected alpha-thalassemia and hemoglobin Constant Spring red blood cells. Am J Clin Pathol. 1988;89: 521-525.
Luzzi GA, Merry AH, Newbold CI, Marsh K, Pasvol G, Weatherall DJ. Surface antigen expression on Plasmodium falciparum-infected erythrocytes is modified in alpha- and beta-thalassemia. J exp Med. 1991;173: 785-791.
Williams TN, Weatherall DJ, Newbold CI. The membrane characteristics of Plasmodium falciparum-infected and -uninfected heterozygous alpha(0)thalassaemic erythrocytes. Br J Haematol. 2002;118: 663-670.
Carlson J, Nash GB, Gabutti V, al-Yaman F, Wahlgren M. Natural protection against severe Plasmodium falciparum malaria due to impaired rosette formation. Blood. 1994;84: 3909-3914.
Mockenhaupt FP, Bienzle U, May J, et al. Plasmodium falciparum infection: influence on hemoglobin levels in alpha-thalassemia and microcytosis. J Infect Dis. 1999;180: 925-928.
Mockenhaupt FP, Falusi AG, May J, et al. The contribution of alpha+-thalassaemia to anaemia in a Nigerian population exposed to intense malaria transmission. Trop Med Int Health. 1999;4: 302-307.
Mockenhaupt FP, May J, Bergqvist Y, Meyer CG, Falusi AG, Bienzle U. evidence for a reduced effect of chloroquine against Plasmodium falciparum in alpha-thalassaemic children. Trop Med Int Health. 2001;6: 102-107.
Williams TN. Mechanisms of malaria protection in the thalassaemia syndromes. Medicine. London, United Kingdom: University of London; 1999.
Chen Q, Schlichtherle M, Wahlgren M. Molecular aspects of severe malaria. Clin Microbiol Rev. 2000;13: 439-450.
Clark IA, Cowden WB, Ba FD. The pathophysiology of falciparum malaria. Pharmacol Ther. 2003;99: 221-260.
Dondorp AM, Kager PA, Vreeken J, White NJ. Abnormal blood flow and red blood cell deformability in severe malaria. Parasitol Today. 2000;16: 228-232.
Menendez C, Fleming AF, Alonso PL. Malaria-related anaemia. Parasitol Today. 2000;16: 469-476.
Maitland K, Newton CR. Acidosis of severe falciparum malaria: heading for a shock? Trends Parasitol. 2005;21: 11-16.
Cockburn IA, Mackinnon MJ, O'Donnell A, et al. A human complement receptor 1 polymorphism that reduces Plasmodium falciparum rosetting confers protection against severe malaria. Proc Natl Acad Sci U S A. 2004;101: 272-277.(Thomas N. Williams, Sammy)
Nuffield Department of Clinical Medicine and Department of Paediatrics, University of Oxford, John Radcliffe Hospital
Neurosciences Unit, Institute of Child Health, University College, London
Department of Paediatrics, Faculty of Medicine and the Wellcome Trust Centre for Clinical Tropical Medicine, Imperial College, London, United Kingdom.
Abstract
Although the + thalassemias almost certainly confer protection against death from malaria, this has not been formally documented. We have conducted a study involving 655 case patients with rigorously defined severe malaria and 648 controls, frequency matched on area of residence and ethnic group. The prevalence of both heterozygous and homozygous + thalassemia was reduced in both case patients with severe malaria (adjusted odds ratios [ORs], 0.73 and 0.57; 95% confidence intervals [95% CIs], 0.57-0.94 and 0.40-0.81; P = .013 and P = .002, respectively, compared with controls) and among the subgroup of children who died after admission with severe malaria (OR, 0.60 and 0.37; 95% CI, 0.37-1.00 and 0.16-0.87; P = .05 and P = .02, respectively, compared with surviving case patients). The lowest ORs were seen for the forms of malaria associated with the highest mortality—coma and severe anemia complicated by deep, acidotic breathing. Our study supports the conclusion that both heterozygotes and homozygotes enjoy a selective advantage against death from Plasmodium falciparum malaria.
Introduction
The + thalassemias are the commonest known genetic disorder in humans,1 a situation that probably reflects a selective advantage against death from malaria. However, to date, the protection they afford against severe and fatal falciparum malaria has been investigated in only 2 studies, and these have yielded conflicting results.2,3 Here we describe a large case-control study in which we have found that both heterozygous and homozygous + thalassemia are associated with a reduced risk for severe and fatal Plasmodium falciparum malaria in children living on the coast of Kenya.
Study design
Case patients with severe malaria and controls
The study was conducted in Kilifi district, where the population is composed predominantly of rural dwellers of the Mijikenda ethnolinguistic group. The epidemiology and clinical characteristics of malaria in the study area have been described in detail previously.4,5 Case patients were children with clinical features of severe P falciparum malaria who were admitted to the high-dependency unit (HDU) at Kilifi District Hospital (KDH), Kenya, between January 2001 and August 2003. We use 3 clinical features to define severe malaria at our hospital: prostration, coma (Blantyre coma score = 2), and deep breathing. At least 1 of these features is present on admission in approximately 98% of our inpatients who die of malaria.6 Children were eligible for the study if they met the following criteria: (1) P falciparum malaria with any of the 3 clinical features of severity; (2) residence within the study area of the Kenya Medical Research Institute (KeMRI) Centre for Geographic Medicine Research, Coast, Kilifi, an area populated by more than 200 000 subjects that has been under continuous demographic surveillance since 20017; and (3) Mijikenda ethnic origin. Children with uncomplicated severe malaria anemia (SMA) (ie, those with P falciparum malaria, in association with a hemoglobin level lower than 5 g/dL but with no signs of respiratory distress) are not routinely treated in our HDU because the inpatient mortality rate in this group is less than 1%.6 As a result, such children were not included as case patients. All case patients were treated according to standard guidelines, as described in detail previously.8 Controls were residents of the same study area who were randomly selected on the basis of ethnic group (Mijikenda) and who were frequency matched to case patients on the basis of location. Controls were younger than 7, but no attempt was made to match on age. The KeMRI National ethical Review Committee approved the study. Individual written informed consent was provided by all study participants or their parents.
Laboratory methods
Routine hematologic and biochemical data are collected on all children admitted to the HDU at KDH, and malaria parasite densities are determined by standard methods, as described previously.8 Case patients and controls were typed for the common African 3.7-kb -globin deletion by polymerase chain reaction (PCR), as described previously,9 using DNA extracted by standard methods (Qiagen DNA Blood Mini Kit; Qiagen, West Sussex, United Kingdom; or Puregene; Gentra Systems, Minneapolis, MN).
Statistical analysis
Categorical data were analyzed using the 2 test for trend, and numerical data were compared using analysis of variance (ANOVA). We determined the odds ratios (ORs) for severe malaria, severe malaria subgroup, and inpatient death by + thalassemia genotype using logistic regression, with and without adjustment for Mijikenda ethnic subgroup, age, and sex.
Results and discussion
Our study included 655 case patients and 648 controls. The + thalassemia gene frequencies in case patients and controls and the presenting characteristics of case patients are summarized in Tables 1, 2, and 3. The prevalence of both heterozygous and homozygous + thalassemia was significantly lower in case patients than in controls, and the lowest ORs were seen for homozygosity in children with the syndromes associated with the highest risk for mortality in our population—symptomatic SMA10 and deep breathing with coma6 (Table 4). Although admission hemoglobin concentrations were similar between genotypic groups (Table 3), the trend toward increasing hemoglobin values across the groups is noteworthy, given that heterozygous and homozygous + thalassemia are associated with anemia at steady state.11 We found no effect of + thalassemia on admission parasite density.
Simultaneous with the showing of an association between homozygous and heterozygous + thalassemia and protection from severe P falciparum disease, we found evidence for a protective association against death from malaria. Among children with severe malaria, admitted to our HDU on the basis of strictly defined clinical criteria, the risk for death in hospital was 40% lower in heterozygotes and more than 60% lower in homozygotes than in healthy children (Table 2). Furthermore, among case patients with fatal disease, severe acidosis (base deficit greater than 15) at admission was less common in homozygotes (1 of 7; 14%) than in healthy persons (17 of 33; 51%) or heterozygotes (14 of 32; 44%), though this trend did not reach statistical significance (2 = 3.5; P = .19).
The genotypic pattern of protection seen in our study was similar to that seen in Papua, New Guinea, where protection was greatest for anemia and acidosis2; however, in the latter study, protection did not reach significance in heterozygotes, and the in-hospital mortality rate was too low (15 of 433; 3.5%) to detect a between-genotype difference.2 Our observations are not, however, in accord with those from Ghana,3 where protection was seen in heterozygotes (OR, 0.74; 95% confidence interval [95% CI], 0.56-0.98; P = .03), but not in homozygotes (OR not reported), and where no protection was seen against in-hospital death despite a mortality rate (11%3) comparable with that seen in our study. These discrepancies may well be attributable to statistical considerations and may be explicable on the basis of the greater power of the current study than previous studies to detect significant differences; however, they may also relate to differences in the design and conduct of these studies. All 3 adopted a case-control design, an approach that is justified on the grounds that severe P falciparum malaria is a relatively rare outcome, affecting, for example, only approximately 10% of 1- to 5-year-olds in the Kilifi study area. Although case-control studies are open to problems of bias and confounding, we believe for several reasons that it is unlikely our results have been affected by such problems. More than 95% of residents of the Kilifi study area are of Mijikenda ancestry. Although this Bantu population can be categorized into 3 main subgroups—Giriama, Chonyi, and Kauma—we have detected no significant differences in the - allele frequencies between these groups in extensive surveys of more than 4000 children (T.N.W., unpublished data, collected between October 2000 and November 2004). Moreover, we adjusted for these ethnic subgroups in our logistic regression analysis, an exercise that made no material difference to our results. Finally, our controls were frequency-matched on location, through our population database, and our analysis was further adjusted for age. Nevertheless, despite these considerations, we cannot exclude the possibility that unknown biases might have affected our study, which should ideally be replicated using an alternative design such as a family-based or a prospective cohort approach.
What then can such studies tell us about the mechanism by which + thalassemia affords protection against malaria? A number of hypotheses have been proposed, including reduced parasite growth and invasion,12 increased phagocytosis of infected red blood cells,12 altered surface antigen expression that potentially augments immunologic clearance,13,14 and decreased parasite rosetting15; nevertheless, for these to be relevant, they must be compatible with observations from epidemiologic studies conducted under conditions of natural malaria transmission. The fact that, in keeping with all previous observations made in vivo,2,3,16-19 we found no effect at the level of parasite density makes it unlikely that + thalassemia protects simply by limiting the growth or increasing the clearance of parasites. Instead, it seems more likely that it acts by attenuating the consequences of malaria infection.
Severe and fatal malaria results from a range of interrelated pathophysiologic processes that affect multiple organ systems.6 These processes are thought to include the local and systemic release of various cytokines, anemia, decreased red cell deformability and adhesion phenomena such as sequestration and rosetting,20-23 and the clinical features of severe malaria (such as coma, acidosis, and hypovolemic shock), likely reflect the balance of such processes within individual patients.24 That + thalassemia protects against a range of clinical manifestations of severe malaria suggests it does so through a mechanism that is central to many of these processes. In this regard, results from a recent study are particularly intriguing. In an extension of the previous study in Papua, New Guinea,2 Cockburn and colleagues,25 made 2 related observations: first, that a promoter polymorphism of red cell complement receptor 1 (CR1), an important receptor for rosetting, was significantly associated with protection from severe falciparum malaria; second, that + thalassemia was independently associated with the reduced expression of red cell CR1. These observations support the earlier work of Carlson,15 who suggested that a number of the hemoglobinopathies might protect against severe malaria by reducing the ability of red cells to form rosettes. Further work will be required to explore these potentially important observations.
Acknowledgements
We thank the study subjects and their families for their cooperation, and we thank the medical, nursing, and support staff who assisted with this study. We thank David Weatherall, Brett Lowe, Norbert Peshu, and David Roberts for advice and support. This paper is published with the permission of the Director of KeMRI.
Footnotes
Prepublished online as Blood First edition Paper, March 15, 2005; DOI 10.1182/blood-2005-01-0313.
Supported by grants (T.N.W., C.R.J.C.N., K.M.) and a program grant (K.M., C.R.J.C.N.) from the Wellcome Trust, UK.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
References
Weatherall DJ, Clegg JB. The thalassaemia syndromes. 3rd ed. Oxford, United Kingdom: Blackwell Scientific Publications; 2002.
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 U S A. 1997;94: 14736-14741.
Mockenhaupt FP, ehrhardt S, Gellert S, et al. (+)Thalassaemia protects from severe malaria in African children. Blood. 2004;104: 2003-2006.
Snow RW, Omumbo JA, Lowe B, et al. Relation between severe malaria morbidity in children and level of Plasmodium falciparum transmission in Africa. Lancet. 1997;349: 1650-1654.
Mbogo CN, Snow RW, Kabiru eW, et al. Low-level Plasmodium falciparum transmission and the incidence of severe malaria infections on the Kenyan coast. Am J Trop Med Hyg. 1993;49: 245-253.
Marsh K, Forster D, Waruiru C, et al. Indicators of life-threatening malaria in African children. N engl J Med. 1995;332: 1399-1404.
Berkley JA, Lowe BS, Mwangi I, et al. Bacteremia among children admitted to a rural hospital in Kenya. N engl J Med. 2005;352: 39-47.
Maitland K, Pamba A, Newton CR, Levin M. Response to volume resuscitation in children with severe malaria. Pediatr Crit Care Med. 2003;4: 426-431.
Chong SS, Boehm CD, Higgs DR, Cutting GR. Single-tube multiplex-PCR screen for common deletional determinants of alpha-thalassemia. Blood. 2000;95: 360-362.
english M, Waruiru C, Amukoye e, et al. Deep breathing in children with severe malaria: indicator of metabolic acidosis and poor outcome. Am J Trop Med Hyg. 1996;55: 521-524.
Williams TN, Maitland K, Ganczakowski M, et al. Red blood cell phenotypes in the alpha + thalassaemias from early childhood to maturity. Br J Haematol. 1996;95: 266-272.
Yuthavong Y, Butthep P, Bunyaratvej A, Fucharoen S, Khurmith S. Impaired parasite growth and increased susceptibility to phagocytosis of Plasmodium falciparum infected alpha-thalassemia and hemoglobin Constant Spring red blood cells. Am J Clin Pathol. 1988;89: 521-525.
Luzzi GA, Merry AH, Newbold CI, Marsh K, Pasvol G, Weatherall DJ. Surface antigen expression on Plasmodium falciparum-infected erythrocytes is modified in alpha- and beta-thalassemia. J exp Med. 1991;173: 785-791.
Williams TN, Weatherall DJ, Newbold CI. The membrane characteristics of Plasmodium falciparum-infected and -uninfected heterozygous alpha(0)thalassaemic erythrocytes. Br J Haematol. 2002;118: 663-670.
Carlson J, Nash GB, Gabutti V, al-Yaman F, Wahlgren M. Natural protection against severe Plasmodium falciparum malaria due to impaired rosette formation. Blood. 1994;84: 3909-3914.
Mockenhaupt FP, Bienzle U, May J, et al. Plasmodium falciparum infection: influence on hemoglobin levels in alpha-thalassemia and microcytosis. J Infect Dis. 1999;180: 925-928.
Mockenhaupt FP, Falusi AG, May J, et al. The contribution of alpha+-thalassaemia to anaemia in a Nigerian population exposed to intense malaria transmission. Trop Med Int Health. 1999;4: 302-307.
Mockenhaupt FP, May J, Bergqvist Y, Meyer CG, Falusi AG, Bienzle U. evidence for a reduced effect of chloroquine against Plasmodium falciparum in alpha-thalassaemic children. Trop Med Int Health. 2001;6: 102-107.
Williams TN. Mechanisms of malaria protection in the thalassaemia syndromes. Medicine. London, United Kingdom: University of London; 1999.
Chen Q, Schlichtherle M, Wahlgren M. Molecular aspects of severe malaria. Clin Microbiol Rev. 2000;13: 439-450.
Clark IA, Cowden WB, Ba FD. The pathophysiology of falciparum malaria. Pharmacol Ther. 2003;99: 221-260.
Dondorp AM, Kager PA, Vreeken J, White NJ. Abnormal blood flow and red blood cell deformability in severe malaria. Parasitol Today. 2000;16: 228-232.
Menendez C, Fleming AF, Alonso PL. Malaria-related anaemia. Parasitol Today. 2000;16: 469-476.
Maitland K, Newton CR. Acidosis of severe falciparum malaria: heading for a shock? Trends Parasitol. 2005;21: 11-16.
Cockburn IA, Mackinnon MJ, O'Donnell A, et al. A human complement receptor 1 polymorphism that reduces Plasmodium falciparum rosetting confers protection against severe malaria. Proc Natl Acad Sci U S A. 2004;101: 272-277.(Thomas N. Williams, Sammy)