0, 95% confidence interval. 0â0.85). Median hemoglobin levels were 1.2 g/dl lower in malaria cases with ... major transmembrane protein of red blood cells and has two ..... Medicine, John Radcliffe Hospital, Headley Way, Headington, Ox-.
Am. J. Trop. Med. Hyg., 60(6), 1999, pp. 1056–1060 Copyright q 1999 by The American Society of Tropical Medicine and Hygiene
PREVENTION OF CEREBRAL MALARIA IN CHILDREN IN PAPUA NEW GUINEA BY SOUTHEAST ASIAN OVALOCYTOSIS BAND 3 STEPHEN J. ALLEN, ANGELA O’DONNELL, NEAL D. E. ALEXANDER, CHARLES S. MGONE, TIM E. A. PETO, JOHN B. CLEGG, MICHAEL P. ALPERS AND DAVID J. WEATHERALL Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, United Kingdom; Papua New Guinea Institute of Medical Research, Goroka, Papua New Guinea
Abstract. Southeast Asian ovalocytosis (SAO) occurs at high frequency in malarious regions of the western Pacific and may afford a survival advantage against malaria. It is caused by a deletion of the erythrocyte membrane band 3 gene and the band 3 protein mediates the cytoadherence of parasitized erythrocytes in vitro. The SAO band 3 variant may prevent cerebral malaria but it exacerbates malaria anemia and may also increase acidosis, a major determinant of mortality in malaria. We undertook a case-control study of children admitted to hospital in a malarious region of Papua New Guinea. The SAO band 3, detected by the polymerase chain reaction, was present in 0 of 68 children with cerebral malaria compared with six (8.8%) of 68 matched community controls (odds ratio 5 0, 95% confidence interval 5 0–0.85). Median hemoglobin levels were 1.2 g/dl lower in malaria cases with SAO than in controls (P 5 0.035) but acidosis was not affected. The remarkable protection that SAO band 3 affords against cerebral malaria may offer a valuable approach to a better understanding of the mechanisms of adherence of parasitized erythrocytes to vascular endothelium, and thus of the pathogenesis of cerebral malaria. with SAO.7 Also, the decreased anion transport of the variant band 3 protein may worsen acidosis which is an important risk factor for mortality in acute malaria.10 We undertook a prospective case-control study of children admitted to Madang hospital with malaria to assess the magnitude of the protection by SAO band 3 against cerebral malaria and to investigate the relationship between the variant and other common severe manifestations of malaria. The frequency of the variant in severe malaria cases was compared with that in controls living in the community. Factors other than SAO band 3 that may contribute to resistance to malaria and may differ between cases and controls include other genetic factors, especially a1-thalassemia, and acquired immunity as a result of the degree of previous exposure to malaria. To minimize confounding by these factors, and local geographic variations in the prevalence of SAO, a community control child was individually-matched to each severe malaria case for age, sex, ethnicity, village, and season.11
Malaria resistance genes provide the best example of natural selection occurring in human populations. Although the mechanisms by which malaria is prevented are not known, recent case-control studies have shown that genetic variants may affect susceptibility to specific severe manifestations of malaria. For example, in African children, HLA-B53 and glucose-6-phosphate dehydrogenase deficiency are associated with protection against both cerebral malaria and severe malarial anemia, whereas a polymorphism of the tumor necrosis factor-a promoter gene increases the risk of cerebral malaria but not malarial anemia.1 Recently, we have reported that homozygous a1-thalassemia affords significant protection against malaria complicated by severe anemia, acidosis and hyperlactatemia in children in Papua New Guinea.2 Southeast Asian ovalocytosis (SAO) occurs in up to 35% of the population of malarious regions of the western Pacific3 and is caused by a deletion of 27 basepairs of the erythrocyte membrane band 3 gene on chromosome 17.4 Band 3 is the major transmembrane protein of red blood cells and has two main functions: the cytoplasmic domain maintains cell shape by attaching the cell membrane to the cell cytoskeleton,5 and the transmembrane domain increases the capacity of the blood to carry carbon dioxide by exchanging intracellular bicarbonate for chloride.6 An isoform of band 3 is present in the distal nephron, in which its anion exchange function contributes to urine acidification.6 The SAO band 3 protein has a deletion of amino acids 400–408 in the boundary between the cytoplasmic and the first transmembrane domains.4 The SAO red blood cells have altered morphology7 and anion transport is reduced to about 40% of normal.8 Homozygotes for SAO band 3 have not been identified and this condition is assumed to be lethal in utero.3 The high frequency of SAO in malarious regions might reflect heterozygote protection against malaria. The SAO band 3 could modify disease caused by malaria in several ways. Protection by SAO band 3 against cerebral malaria was suggested in a clinical study of children from rural areas of Madang,9 although the possibility of confounding by other factors that reduce malarial disease was not considered in this study. We have reported that malaria anemia is exacerbated in children
MATERIALS AND METHODS
Study site and population. The study was undertaken between October 1993 and February 1996 and details of the study site and population have been published previously.2 The study was based in the pediatric ward of Madang Hospital, which is situated on the north coast of Papua New Guinea, a region hyperendemic for Plasmodium falciparum.12 Only children who had lived for at least 12 months in Madang were included in the study. Ethnicity was categorized according to the region of origin of the languages spoken by children’s parents. The study was approved by the Medical Research Advisory Committee of Papua New Guinea and consent for inclusion was obtained from accompanying parents. The relationship between band 3 status and the risk of death caused by malaria was assessed in two ways. First, in children admitted to hospital with malaria, indices of the severity of disease were compared according to band 3 status. Second, cases with one or more severe manifestations
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of malaria, defined according to World Health Organization criteria,13 were selected as surrogates for children who would be expected to die of malaria. Band 3 status in these index cases was compared with that in individually matched community controls. Definitions of severe malaria. Cerebral malaria was defined as a Blantyre coma score13 # 2 in children with asexual P. falciparum parasitemia and no evidence of bacterial or viral meningoencephalitis on examination of cerebrospinal fluid. The level of consciousness was determined $ 30 min after a generalized convulsion and $ 6 hr after anticonvulsant treatment. In children with asexual P. falciparum parasitemia, severe malarial anemia was defined as a febrile illness with a hemoglobin level , 5g/dl,2 acidosis as a plasma bicarbonate concentration , 15 mmol/L, and hyperlactatemia as a plasma lactate concentration $ 5mmol/L. Recruitment of community controls. The house of the index case was visited soon after the child’s admission and a neighboring house of an unrelated family was selected randomly by spinning a pencil. One child matched as closely as possible to the index case for ethnicity, age (less than one year difference), and sex was selected. If no suitable child was available, the next house in a clockwise direction was visited. Other clinical groups. Protection against mild malarial disease was assessed by comparing band 3 status in the community controls with that in children treated for malaria (fever and P. falciparum parasitemia $ 10,000/ml) as out-patients in six local clinics. The relationship between the variant and the prevalence of malaria in the community was assessed by comparing malariometric indices in the community control children according to band 3 status. To assess the specificity of any protection against malaria detected in the preceding analyses, the frequency of SAO band 3 in the community controls was compared with that in non-malaria controls with severe illness (hospital cases with mostly acute infections but without parasitemia) and mild illness (febrile children without parasitemia treated as out-patients). Laboratory methods. Blood count (MD8; Coulter Electronics, Luton, United Kingdom), biochemical indices (dry slide chemistry, Ektachem; Eastman Kodak, Rochester, NY) and glucose-6-phosphate dehydrogenase activity (procedure no. 400; Sigma, St. Louis, MO) were measured in venous blood. The P. falciparum density was calculated from the number of parasites per 200 white blood cells in thick blood films stained with Giemsa and the measured white blood cell count. A DNA lysate was prepared from heparinized blood and SAO band 3 was detected by the polymerase chain reaction4 and the genotype for a1-thalassemia by Southern blotting.14 In hospital cases, urine was collected into a sterile container, adhesive bags (Urogard; Terumo Corporation, Tokyo, Japan) or post-mortem by supra-pubic aspiration, and tested with Combur7 Test strips (Boehringer Mannheim, Lewes, United Kingdom). The determination of severe manifestations of malaria and the categorization of patients into the various clinical groups was completed before results of the genetic analyses were known. Statistical analysis. Categorical variables were analyzed by the chi-square test and continuous variables by the Kruskal-Wallis test. Odds ratios were derived from the index case–community control pairs who were discordant for band
TABLE 1 Frequency of the Southeast Asian ovalocytosis (SAO) band 3 variant according to clinical group and risk of clinical disease compared with community controls* SAO band 3
Risk of clinical disease for SAO band 3
Clinical group
n
(%)
OR (95% CI)
P
All hospital malaria Clinic malaria Hospital non-malaria Clinic non-malaria Community controls Total
27/431 7/110 14/240 7/136 18/307 73/1,224
(6.3) (6.4) (5.8) (5.1) (5.9) (6.0)
1.07 (0.58–1.99) 1.09 (0.44–2.69) 1.0 (0.48–2.04) 0.87 (0.36–2.14) – –
0.82 0.85 0.99 0.76
* OR 5 odds ratio; CI 5 confidence interval.
3 status and 95% confidence intervals for the odds ratios were based on the exact confidence interval for a binomial proportion.15 Possible confounding caused by differences between cases and controls in ethnicity and age, which occurred despite matching, and genotype for a1-thalassemia, was assessed by including these variables in a conditional logistic regression analysis11 in which case-control status was the dependent variable and band 3 status the predictor variable. Comparison of band 3 status in the community controls with that in other clinical groups was by logistic regression analysis. EGRET software (Statistics and Epidemiology Research Corp., Seattle, WA) was used for all regression analyses. RESULTS
Sufficient DNA was available from 1,224 (81.4%) of 1,503 children to determine SAO band 3 status. The frequency of SAO was 6.8% (70 of 1,023) in children with one or both parents of Madang ethnicity, 1.4% (2 of 142) in Sepiks, 3.0% (1 of 33) in individuals from other coastal regions, and 0% in 26 Highlanders. The frequency was 8.1% (27 of 334) in a1-thalassemia homozygotes, 8.0% (23 of 288) in heterozygotes and 3.5% (5 of 142) in children without a1-thalassemia (P 5 0.17). Among boys, glucose-6phosphate dehydrogenase deficiency was detected in 0 of 30 children with the variant and in 15 (2.9%) of 522 children with a normal band 3 (P 5 1.00, by Fisher’s exact test). Frequency of SAO band 3 in the clinical groups. The SAO band 3 was present in 5.9% of 307 community controls, and a similar frequency was observed in all children admitted to the hospital with malaria, in children treated for malaria as out-patients, and in controls with severe and mild non-malarial illnesses (Table 1). Logistic regression analysis showed that band 3 status was not significantly associated with the risk of clinical disease, including when differences in the frequency of demographic variables and genotype for a1-thalassemia between some of the clinical groups and the community controls2 were included in the model. Cerebral malaria. Among the hospital malaria cases, cerebral malaria was diagnosed in 17.6% of the children with a normal band 3 but in none of 27 children with an SAO band 3 (P 5 0.013; Table 2). There were 284 severe malaria–community control pairs (Table 3). The SAO band 3 was present in six of 68 community controls selected for the cerebral malaria cases and conditional logistic regression
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TABLE 2 Laboratory variables, Blantyre coma score, and outcome according to band 3 status in all children admitted to hospital with malaria* Band 3 genotype Normal Variable
Laboratory Hemoglobin (g/dl) Glucose (mmol/l) Bicarbonate (mmol/l) Lactate (mmol/l) Plasmodium falciparum density (log10 parasites/ml)
Median
(IQR)
n
Median
(IQR)
404 384 377 375
6.0 4.9 21 2.9
(4.3–9.1) (4.1–5.6) (17–24) (2.0–4.6)
27 25 25 25
4.8 4.9 20 3.0
(3.9–7.0) (4.5–5.6) (17–22) (2.4–4.2)
0.035 0.40 0.29 0.60
4.3
(3.4–4.9)
(3.7–4.8)
0.69
388
Urine pH 5 or 6 no. (%) 279/322 Coma score no. (%) #2 (cerebral malaria) 70/398 #4 (impaired consciousness) 86/398 Outcome Died or neurologic sequelae
SAO deletion
n
P
25
4.0
(86.7)
17/23
(73.9)
(17.6) (21.6)
0/27 0/27
(0) (0)
0.013† 0.0025†
(5.0)
0/27
(0)
0.63†
0.12†
no. (%) 20/404
* Data are number and median value (inter-quartile range [IQR]) unless otherwise noted. Numbers vary because of missing data. Continuous variables were analyzed by the Kruskal-Wallis test and categorical variables by the chi-square test. SAO 5 Southeast Asian ovalocytosis. † By Fisher’s exact test.
with a normal band 3 had acidic urine (P 5 0.60, by Fisher’s exact test).
analysis showed that the variant reduced significantly the risk of cerebral malaria (odds ratio 5 0, 95% confidence interval 5 0–0.85, Table 3). Including age, ethnicity, and genotype for a1-thalassemia in the regression model did not materially alter the results. Anemia. In 431 children admitted to the hospital with malaria, median hemoglobin levels were significantly lower in children with SAO band 3 than in those with a normal band 3 (Table 2). In the 173 case-control pairs in which the case had severe anemia, the increased frequency of the variant in the cases compared with their controls was of borderline statistical significance (Table 3). Median hemoglobin levels and malariometric indices in community control children were similar in those with and without SAO band 3 (Table 4). Acidosis. Plasma bicarbonate did not vary according to band 3 status in the hospital malaria cases (Table 2) and SAO did not prevent admission with acidosis in the casecontrol analysis (Table 3). Urine pH varied from 5 to 9 and acidic urine (pH 5 or 6) was present in a similar proportion of cases with SAO band 3 and those with a normal band 3 (Table 2). Similarly, in the non-malaria hospital cases, all 10 children with variant band 3 and 117 (87%) of 135 children
DISCUSSION
The SAO band 3 has now been shown to protect against cerebral malaria in two prospective case-control studies, each using different controls. Previously, the variant band 3 was absent in 35 cerebral malaria cases but was present in 15 of 103 population controls (P 5 0.01).9 In the present study, community controls were matched individually to index cases to account for local geographic variations in the prevalence of SAO and to minimize differences between cases and controls in other factors that mediate immunity to malaria. The recruitment of cerebral malaria cases in the present study overlapped with that in the previous study such that one case was common to both studies. When the data from the two studies are combined (ignoring the case-control matching in the present study), SAO band 3 appears to confer remarkable protection against cerebral malaria (MantelHaenszel summary x2 5 9.79, P 5 0.0018, odds ratio 5 0, exact upper 95% confidence limit16 5 0.35), indicating a
TABLE 3 Risk of developing severe malaria according to band 3 status in the index case-community control pairs* Number of discordant pairs SAO band 3 present Clinical group
Case
Control
Odds ratio (95% CI)
All severe malaria (total no. of pairs, n 5 284)
19
16
1.19 (0.58–2.47)
0.61
Clinical subgroups Severe anemia (n 5 173) Cerebral malaria (n 5 68) Acidosis (n 5 52) Hyperlactatemia (n 5 66)
15 0 3 4
7 6 1 1
2.14 (0.82–6.21) 0 (0–0.85) 3.00 (0.24–157) 4.00 (0.40–197)
0.084 0.031 0.31 0.17
P
* Both members were of Madang ethnicity in 226 pairs. In the majority of pairs, both members did not have Southeast Asian ovalocytosis (SAO) band 3 and there were no pairs in which both members had the variant. Case-control pairs that were discordant for SAO band 3 are shown in the table. The odds ratios represent the risk of developing severe malaria for children with SAO band 3 relative to normals. CI 5 confidence interval.
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TABLE 4 Hemoglobin and malariometric indices according to band 3 status in community control children* Band 3 genotype Variable
Normal
Hemoglobin (g/dl) Plasmodium falciparum rate density/ml (log10 value) P. vivax rate density/ml (log10 value)
10.0 (8.8–11.0) [283] 114/284 (40.1) 3.1 (2.5–3.8) 45/284 (15.9) 2.5 (2.1–3.0)
10.1 (8.8–10.8) [15] 6/18 (33.3) 3.0 (2.6–3.1) 2/18 (11.1) 3.5 (3.4–3.7)
SAO deletion
0.75 0.75 0.55 0.84 0.082
P
Spleen (Hackett grade) 0 1–3 4–5
156 (54.0) 122 (42.2) 11 (3.8)
10 (55.6) 6 (33.3) 2 (11.1)
0.22
* Continuous variables are shown as median value (inter-quartile range) [n] and categorical variables as number (%). Numbers vary because of missing data. Chi-square test for parasite rates, chi-square test for trend for splenomegaly, and Kruskal-Wallis test for other variables. SAO 5 Southeast Asian ovalocytosis.
protective effect equal to, or perhaps greater than, that afforded by the sickle cell trait.17 Cerebral malaria was associated with an 8% case fatality in this series,10 but case fatality rates of up to 40% have been reported in African children.13 The sequestration of parasitized red blood cells in the cerebral vasculature is thought to be central to the pathogenesis of cerebral malaria18 and band 3 has been implicated in this process. Plasmodium falciparum infection of red blood cells results in a modification of band 3 such that two normally cryptic amino acid sequences, consisting of residues 547–553 and 829–824, are expressed on the red blood cell surface.19 Synthetic peptides based on these sequences inhibited adherence of infected erythrocytes to melanoma cells in vitro and appeared to prevent sequestration when infused into P. falciparum-infected Aotus and Saimiri monkeys.20 The deletion of amino acids 400–408 in SAO band 3 markedly increases the rigidity of the red blood cell membrane21 and may prevent the expression of the cytoadherent sequences. Also, in view of the reduced expression of several blood group antigens22 in ovalocytic red blood cells, the expression of other red blood cell cytoadherence ligands might also be reduced. We have reported previously that malaria anemia is exacerbated in SAO and that this may be caused partly by the selective removal of ovalocytic red blood cells by the spleen.7 Severe anemia was the most common severe manifestation of malaria in this series, and although not predictive of mortality during the hospital admission,10 it may contribute to indirect malaria mortality in the community. Acidosis (plasma bicarbonate , 15mmol/L) occurred in 12.7% of the hospital malaria cases in this series and was associated with a case fatality rate of 17.2%.10 Whether the anion exchange function of the erythrocyte band 3 protein, and its isoform in the kidney, is essential for normal acidbase homeostasis is not known. A normal ability to acidify urine has been demonstrated in one individual with SAO,23 but this has not been studied during acute illness. Mutations in the band 3 gene responsible for hereditary spherocytosis reduce anion transport but impairment of urinary acidification has not been reported.24 Familial distal renal tubular acidosis is associated with mutations in band 3, but the resultant decrease in anion transport is not thought to be sufficient to account for the reduced acid-secreting ability of the renal tubular cells.23,25 Total deficiency of red blood cell band 3 occurs in Japanese black cattle and is associated with
a high neonatal death rate.26 However, among surviving animals, the plasma bicarbonate level was reduced to only 75% of normal ones, gas exchange in the lungs was not impaired and hydrogen ion excretion by the kidney remained effective.26 Our findings that plasma bicarbonate levels in hospital malaria cases and urine pH in hospital malaria and nonmalaria cases did not vary according to band 3 status suggest that other anion exchange channels compensate for the reduced anion transport of the variant band 3 protein even during severe illness. The similar frequency of SAO band 3 in the non-malaria hospital cases as in the community controls suggests that it does not reduce indirect malaria mortality or mortality caused by non-malarial infections. Also, the band 3 variant did not appear to prevent mild disease caused by either malaria or non-malarial infections. Although invasion of ovalocytic red blood cells by both P. falciparum and P. knowlesi has been reported to be reduced in vitro,27,28 cross-sectional studies undertaken in Madang have given conflicting results regarding the protection that SAO provides against parasitemia.7 In the present study, the similar rate and density of both P. falciparum and P. vivax parasitemia in the community controls with and without SAO band 3 suggests that it offers no protection against malaria parasitemia. Southeast Asian ovalocytosis is a common red blood cell surface variant in malarious regions of the western Pacific. Its selective advantage appears to be conferred by its powerful protection against cerebral malaria. Investigation of the adhesion properties of parasitized red blood cells with the variant may shed light on the molecular pathogenesis of this severe manifestation of malaria. The selective advantage of SAO is balanced by an exacerbation of malaria anemia in heterozygotes and nonviability of the homozygote state. Acknowledgments: We are indebted to the people of Madang for participating in this study. We thank W. Deppsone for meticulous field work, J. J. Martinson, M. Mellombo, and A. Raiko for laboratory assistance, and Professor D. A. Warrell for advice and help in setting up this project. Professor M. J. A. Tanner also gave useful advice. Financial support: This work was supported by the Wellcome Trust and the Medical Research Council. Authors’ addresses: Stephen J. Allen, Angela O’Donnell, Tim E. A. Peto, John B. Clegg, and David J. Weatherall, Institute of Molecular Medicine, John Radcliffe Hospital, Headley Way, Headington, Oxford, OX3 9DS, United Kingdom. Neal D. E. Alexander and Charles
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S. Mgone and Michael P. Alpers, Papua New Guinea Institute of Medical Research, PO Box 60, Goroka, EHP 441, Papua New Guinea.
16.
Reprint requests: Stephen J. Allen, Medical Research Council Laboratories, Fajara, PO Box 273, Banjul, The Gambia.
17.
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