• View in gallery

    Age-specific levels of malaria antibodies in children with or without alpha+-thalassemia versus without, adjusted for parasite density. A, Log-transformed arbitrary units of mean fluorescence intensities (MFIs) of accumulated IgG antibodies with specificity for variant surface antigens on six heterologous P. falciparum parasite isolates. B, Log-transformed arbitrary units of OD values of IgG antibody response with specificity to recombinant GLURP-R0 antigen. Data presented are from April 2001. Normal, αα/αα; alpha +-thalassemia, αα/α− or α−/α−. Age groups in years: 0–3, 3–5, and 5–19. Number of children in each group: see Table 3. Box plots show medians with 25th and 75th percentiles, whiskers for 10th and 90th percentiles, and dots for outliers. *Higher levels of anti-GLURP IgG, but not significantly, in children with alpha+-thalassemia 3–5 years of age as calculated by the Student unpaired t test.

  • 1

    Mcgregor IA, 1987. Malarial immunity—current trends and prospects. Ann Trop Med Parasitol 81 :647–656.

  • 2

    Allison AC, 1964. Polymorphism and natural selection in human populations. Cold Spring Harb Symp Quant Biol 29 :137–149.

  • 3

    Flint J, Hill AVS, Bowden DK, Oppenheimer SJ, Sill PR, Serjeantson SW, Banakoiri J, Bhatia K, Alpers MP, Boyce AJ, Weatherall DJ, Clegg JB, 1986. High-frequencies of alpha-thalassemia are the result of natural-selection by malaria. Nature 321 :744–750.

    • Search Google Scholar
    • Export Citation
  • 4

    Ruwende C, Khoo SC, Snow AW, Yates SNR, Kwiatkowski D, Gupta S, Warn P, Allsopp CEM, Gilbert SC, Peschu N, Newbold CI, Greenwood BM, Marsh K, Hill AVS, 1995. Natural-selection of hemizygotes and heterozygotes for G6PD deficiency in Africa by resistance to severe malaria. Nature 376 :246–249.

    • Search Google Scholar
    • Export Citation
  • 5

    Beutler E, 1991. Glucose-6-phosphate-dehydrogenase deficiency. N Engl J Med 324 :169–174.

  • 6

    Weatherall DJ, Clegg JB, 2001. Inherited haemoglobin disorders: an increasing global health problem. Bull WHO 79 :704–712.

  • 7

    Aidoo M, Terlouw DJ, Kolczak M, McElroy PD, ter Kuile FO, Kariuki S, Nahlen BL, Lal AA, Udhayakumar V, 2002. Protective effects of the sickle cell gene against malaria morbidity and mortality. Lancet 359 :1311–1312.

    • Search Google Scholar
    • Export Citation
  • 8

    Mockenhaupt FP, Ehrhardt S, Gellert S, Otchwemah RN, Dietz E, Anemana SD, Bienzle U, 2004. alpha(+)-thalassemia protects African children from severe malaria. Blood 104 :2003–2006.

    • Search Google Scholar
    • Export Citation
  • 9

    Williams TN, Wambua S, Uyoga S, Macharia A, Mwacharo JK, Newton CRJC, Maitland K, 2005. Both heterozygous and homozygous alpha(+) thalassemias protect against severe and fatal Plasmodium falciparum malaria on the coast of Kenya. Blood 106 :368–371.

    • Search Google Scholar
    • Export Citation
  • 10

    Allison AC, 1954. Protection afforded by sickle-cell trait against subtertian malarial infection. BMJ 1 :290–294.

  • 11

    Hill AVS, Allsopp CEM, Kwiatkowski D, Anstey NM, Twumasi P, Rowe PA, Bennett S, Brewster D, Mcmichael AJ, Greenwood BM, 1991. Common West African Hla antigens are associated with protection from severe malaria. Nature 352 :595–600.

    • Search Google Scholar
    • Export Citation
  • 12

    Williams TN, Mwangi TW, Wambua S, Alexander ND, Kortok M, Snow RW, Marsh K, 2005. Sickle cell trait and the risk of Plasmodium falciparum malaria and other childhood diseases. J Infect Dis 192 :178–186.

    • Search Google Scholar
    • Export Citation
  • 13

    Guindo A, Fairhurst RM, Doumbo O, Wellems TE, Diallo D, 2007. X-linked G6PD deficiency protects hemizygous males but not heterozygous females against severe malaria. PLoS Med 4 :e66.

    • Search Google Scholar
    • Export Citation
  • 14

    Wambua S, Mwangi TW, Kortok M, Uyoga S, Macharia AW, Mwacharo JK, Weatherall DJ, Snow RW, Marsh K, Williams TN, 2006. The effect of alpha+ thalassaemia on the incidence of malaria and other diseases in children living on the coast of Kenya. PLoS Med 3 :e158.

    • Search Google Scholar
    • Export Citation
  • 15

    Williams TN, Maitland K, Bennett S, Ganczakowski M, Peto TEA, Newbold CI, Bowden DK, Weatherall DJ, Clegg JB, 1996. High incidence of malaria in alpha-thalassaemic children. Nature 383 :522–525.

    • Search Google Scholar
    • Export Citation
  • 16

    Oppenheimer SJ, Hill AVS, Gibson FD, Macfarlane SB, Moody JB, Pringle J, 1987. The interaction of alpha-Thalassemia with malaria. Trans R Soc Trop Med Hyg 81 :322–326.

    • Search Google Scholar
    • Export Citation
  • 17

    Roth EF, Raventossuarez C, Rinaldi A, Nagel RL, 1983. Glucose-6-phosphate-dehydrogenase deficiency inhibits in vitro growth of Plasmodium falciparum.Proc Natl Acad Sci USA 80 :298–299.

    • Search Google Scholar
    • Export Citation
  • 18

    Pasvol G, Weatherall DJ, Wilson RJM, 1978. Cellular mechanism for protective effect of haemoglobin-S against P. falciparum malaria. Nature 274 :701–703.

    • Search Google Scholar
    • Export Citation
  • 19

    Cappadoro M, Giribaldi G, O’Brien E, Turrini F, Mannu F, Ulliers D, Simula G, Luzzatto L, Arese P, 1998. Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum may explain malaria protection in G6PD deficiency. Blood 92 :2527–2534.

    • Search Google Scholar
    • Export Citation
  • 20

    Ayi K, Turrini F, Piga A, Arese P, 2004. 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 104 :3364–3371.

    • Search Google Scholar
    • Export Citation
  • 21

    Luzzi GA, Merry AH, Newbold CI, Marsh K, Pasvol G, 1991. Protection by alpha-thalassemia against Plasmodium falciparum malaria—modified surface-antigen expression rather than impaired growth or cytoadherence. Immunol Lett 30 :233–240.

    • Search Google Scholar
    • Export Citation
  • 22

    Marsh K, Otoo L, Hayes RJ, Carson DC, Greenwood BM, 1989. Antibodies to blood stage antigens of Plasmodium falciparum in rural Gambians and their relation to protection against infection. Trans R Soc Trop Med Hyg 83 :293–303.

    • Search Google Scholar
    • Export Citation
  • 23

    Pasvol G, 1992. A cellular mechanism for the protection by thalassemia against Plasmodium falciparum malaria. Br J Haematol 82 :267.

  • 24

    Bayoumi RA, Abuzeid YA, Abdulhadi NH, Saeed BO, Theander TG, Hviid L, Ghalib HW, Nugud AHD, Jepsen S, Jensen JB, 1990. Cell-mediated immune-responses to Plasmodium falciparum purified soluble antigens in sickle-cell trait subjects. Immunol Lett 25 :243–250.

    • Search Google Scholar
    • Export Citation
  • 25

    Arie T, Fairhurst RM, Brittain NJ, Wellems TE, Dvorak JA, 2005. Hemogloblin C modulates the surface topography of Plasmodium falciparum-infected erythrocytes. J Struct Bio 150 :163–169.

    • Search Google Scholar
    • Export Citation
  • 26

    Verra F, Simpore J, Warimwe GM, Tetteh KK, Howard TOF, Bancone G, Avellino P, Blot I, Fegan G, Bull PC, Williams TN, Conway DJ, Marsh K, Modiano D, 2007. Haemoglobin C and S role in acquired immunity against Plasmodium falciparum malaria. PloS One e978.

    • Search Google Scholar
    • Export Citation
  • 27

    Cabrera G, Cot M, Migot-Nabias F, Kremsner PG, Deloron P, Luty AJF, 2005. The sickle cell trait is associated with enhanced immunoglobulin G antibody responses to Plasmodium falciparum variant surface antigens. J Infect Dis 191 :1631–1638.

    • Search Google Scholar
    • Export Citation
  • 28

    Jakobsen PH, Riley EM, Allen SJ, Larsen SO, Bennett S, Jepsen S, Greenwood BM, 1991. Differential antibody-response of Gambian donors to soluble Plasmodium falciparum antigens. Trans R Soc Trop Med Hyg 85 :26–32.

    • Search Google Scholar
    • Export Citation
  • 29

    Allen SJ, Bennett S, Riley EM, Rowe PA, Jakobsen PH, Odonnell A, Greenwood BM, 1992. Morbidity from malaria and immune-responses to defined Plasmodium falciparum antigens in children with sickle-cell trait in the Gambia. Trans R Soc Trop Med Hyg 86 :494–498.

    • Search Google Scholar
    • Export Citation
  • 30

    Le Hesran JY, Personne I, Personne P, Fievet N, Dubois B, Beyeme M, Boudin C, Cot M, Deloron P, 1999. Longitudinal study of Plasmodium falciparum infection and immune responses in infants with or without the sickle cell trait. Int J Epidemiol 28 :793–798.

    • Search Google Scholar
    • Export Citation
  • 31

    Maya DWM, Mavoungou E, Deloron P, Theisen M, Ntoumi F, 2006. Distribution of IgG subclass antibodies specific for Plasmodium falciparum glutamate-rich-protein molecule in sickle cell trait children with asymptomatic infections. Exp Parasitol 112 :92–98.

    • Search Google Scholar
    • Export Citation
  • 32

    Ntoumi F, Ekala MT, Makuwa M, Lekoulou F, Mercereau-Puijalon O, Deloron P, 2002. Sickle cell trait carriage: imbalanced distribution of IgG subclass antibodies reactive to Plasmodium falciparum family-specific MSP2 peptides in serum samples from Gabonese children. Immunol Lett 84 :9–16.

    • Search Google Scholar
    • Export Citation
  • 33

    Sarr JB, Pelleau S, Toly C, Guitard J, Konate L, Deloron P, Garcia A, Migot-Nabias F, 2006. Impact of red blood cell polymorphisms on the antibody response to Plasmodium falciparum in Senegal. Microbes Infect 8 :1260–1268.

    • Search Google Scholar
    • Export Citation
  • 34

    Lusingu JPA, Vestergaard LS, Mmbando BP, Drakeley CJ, Jones C, Akida J, Savaeli ZX, Kitua AY, Lemnge MM, Theander TG, 2004. Malaria morbidity and immunity among residents of villages with different Plasmodium falciparum transmission intensity in North-Eastern Tanzania. Malar J 3 :26.

    • Search Google Scholar
    • Export Citation
  • 35

    Pearce RJ, Drakeley C, Chandramohan D, Mosha F, Roper C, 2003. Molecular determination of point mutation haplotypes in the dihydrofolate reductase and dihydropteroate synthase of Plasmodium falciparum in three districts of northern Tanzania. Antimicrob Agents Chemo 47 :1347–1354.

    • Search Google Scholar
    • Export Citation
  • 36

    Enevold A, Vestergaard LS, Lusingu J, Drakeley CJ, Lemnge MM, Theander TG, Bygbjerg IC, Alifrangis M, 2005. Rapid screening for glucose-6-phosphate dehydrogenase deficiency and haemoglobin polymorphisms in Africa by a simple high-throughput SSOP-ELISA method. Malar J 4 :61.

    • Search Google Scholar
    • Export Citation
  • 37

    Liu YT, Old JM, Miles K, Fisher CA, Weatherall DJ, Clegg JB, 2000. Rapid detection of alpha-thalassaemia deletions and alpha-globin gene triplication by multiplex polymerase chain reactions. Br J Haematol 108 :295–299.

    • Search Google Scholar
    • Export Citation
  • 38

    Lusingu JPA, Vestergaard LS, Alifrangis M, Mmbando BP, Theisen M, Kitua AY, Lemnge MM, Theander TG, 2005. Cytophilic antibodies to Plasmodium falciparum glutamate rich protein are associated with malaria protection in an area of holoendemic transmission. Malar J 4 :48.

    • Search Google Scholar
    • Export Citation
  • 39

    Staalsoe T, Giha HA, Dodoo D, Theander TG, Hviid L, 1999. Detection of antibodies to variant antigens on Plasmodium falciparum- infected erythrocytes by flow cytometry. Cytometry 35 :329–336.

    • Search Google Scholar
    • Export Citation
  • 40

    Mockenhaupt FP, Falusi AG, May J, Ademowo OG, Olumese PE, Meyer CG, Bienzle U, 1999. The contribution of alpha(+)-thalassaemia to anaemia in a Nigerian population exposed to intense malaria transmission. Trop Med Int Health 4 :302–307.

    • Search Google Scholar
    • Export Citation
  • 41

    Modiano G, Morpurgo G, Terrenato L, Novelletto A, Dirienzo A, Colombo B, Purpura M, Mariani M, Santachiarabenerecetti S, Brega A, Dixit KA, Shrestha SL, Lania A, Wanachiwanawin W, Luzzatto L, 1991. Protection against malaria morbidity—near-fixation of the alpha-thalassemia gene in a Nepalese population. Am J Hum Genet 48 :390–397.

    • Search Google Scholar
    • Export Citation
  • 42

    Allen SJ, O’Donnell A, Alexander NDE, Alpers MP, Peto TEA, Clegg JB, Weatherall DJ, 1997. Alpha(+)-thalassemia protects children against disease caused by other infections as well as malaria. Proc Natl Acad Sci USA 94 :14736–14741.

    • Search Google Scholar
    • Export Citation
  • 43

    Williams TN, Mwangi TW, Roberts DJ, Alexander ND, Weatherall DJ, Wambua S, Kortok M, Snow RW, Marsh K, 2005. An immune basis for malaria protection by the sickle cell trait. PLoS Med 2 :441–445.

    • Search Google Scholar
    • Export Citation
  • 44

    Dodoo D, Theisen M, Kurtzhals JAL, Akanmori BD, Koram KA, Jepsen S, Nkrumah FK, Theander TG, Hviid L, 2000. Naturally acquired antibodies to the glutamate-rich protein are associated with protection against Plasmodium falciparum malaria. J Infect Dis 181 :1202–1205.

    • Search Google Scholar
    • Export Citation
  • 45

    Bull PC, Lowe BS, Kortok M, Molyneux CS, Newbold CI, Marsh K, 1998. Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria. Nat Med 4 :358–360.

    • Search Google Scholar
    • Export Citation
  • 46

    Oeuvray C, Theisen M, Rogier C, Trape JF, Jepsen S, Druilhe P, 2000. Cytophilic immunoglobulin responses to Plasmodium falciparum glutamate-rich protein are correlated with protection against clinical malaria in Dielmo, Senegal. Infect Immun 68 :2617–2620.

    • Search Google Scholar
    • Export Citation
  • 47

    Marsh K, Howard RJ, 1986. Antigens induced on erythrocytes by P. falciparum: expression of diverse and conserved determinants. Science 231 :150–153.

    • Search Google Scholar
    • Export Citation
  • 48

    Bull PC, Marsh K, 2002. The role of antibodies to Plasmodium falciparum-infected-erythrocyte surface antigens in naturally acquired immunity to malaria. Trends Microbiol 10 :55–58.

    • Search Google Scholar
    • Export Citation
  • 49

    Urban BC, Shafi MJ, Cordery DV, Macharia A, Lowe B, Marsh K, Williams TN, 2006. Frequencies of peripheral blood myeloid cells in healthy Kenyan children with alpha+ thalassemia and the sickle cell trait. Am J Trop Med Hyg 74 :578–584.

    • Search Google Scholar
    • Export Citation
  • 50

    Enevold A, Alifrangis M, Sanchez J, Carneiro I, Roper C, Børsting C, Lusingu J, Vestergaard L, Lemnge M, Morling N, Riley E, Drakeley C, 2007. Associations between alpha +-Thalassemia and Plasmodium falciparum Malarial Infection in Northeastern Tanzania. J Infect Dis 196 :451–459.

    • Search Google Scholar
    • Export Citation
  • 51

    Williams TN, Mwangi TW, Wambua S, Peto TEA, Weatherall DJ, Gupta S, Recker M, Penman BS, Uyoga S, Macharia A, Mwacharo JK, Snow RW, Marsh K, 2005. Negative epistasis between the malaria-protective effects of alpha(+)-thalassemia and the sickle cell trait. Nat Gen 37 :1253–1257.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Reduced Risk of Uncomplicated Malaria Episodes in Children with Alpha+-Thalassemia in Northeastern Tanzania

View More View Less
  • 1 Centre for Medical Parasitology, Institute for International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark; National Institute for Medical Research, Tanga Medical Research Centre, Tanga, Tanzania; Department of Epidemiology, Statens Serum Institute, Copenhagen, Denmark

The prevalence of human red blood cell (RBC) polymorphisms is high in areas of intense Plasmodium falciparum transmission, and individuals carrying these genetic traits are believed to be partially protected against severe malaria. However, it remains uncertain how RBC polymorphisms affect the susceptibility to uncomplicated malaria. We compared the risk of suffering from febrile, uncomplicated malaria between individuals carrying three common RBC polymorphisms (sickle cell trait, alpha+-thalassemia, and glucose-6-phosphate-dehydrogenase deficiency) and controls. The study was performed in an area of intense malaria transmission where 202 individuals 0–19 years of age were monitored clinically for a period of 6 months. RBC polymorphisms were assessed with molecular methods, and plasma antibodies to P. falciparum variant surface antigens (anti-VSA IgG) and glutamate-rich protein (anti-GLURP IgG) were measured with flow cytometry and ELISA assays, respectively. Regression analyses showed that alpha+-thalassemia was associated with a reduced risk of uncomplicated malaria episodes and that this advantageous effect seemed to be more predominant in children older than 5 years of age, but was independent of levels of antibodies to VSA and GLURP.

INTRODUCTION

Individuals living in malaria-endemic areas, repeatedly exposed to Plasmodium falciparum infections, are partially protected from clinical malaria episodes by various mechanisms of acquired immunity.1 In addition, a variety of inherited genetic traits, conferring a level of innate immunity, has been selected in populations under high malaria pressure.24 The most studied traits are the human red blood cell (RBC) polymorphisms, of which structural hemoglobin variants (HbS, HbC, and HbE), thalassemias (α and β), and glucose-6-phosphate-dehydrogenase (G6PD) deficiency are widespread in present or former malaria-endemic areas.5,6 These traits have all been found to be associated with reduced susceptibility to severe malaria.4,79 Individuals with sickle cell trait (HbAS) also seem to be protected against uncomplicated malaria and parasitemia,1012 but data for the relationship between uncomplicated malaria and alpha+-thalassemia or G6PD deficiency are conflicting. Some studies have reported that the incidence of uncomplicated malaria is unaltered or slightly reduced,4,13,14 whereas other studies have indicated that the incidence is increased.15,16

It remains largely unknown how the different RBC polymorphisms affect malaria susceptibility. Among suggested effector mechanisms are impairment of parasite invasion and growth of RBCs,17,18 enhanced phagocytosis and premature removal of infected RBC,19,20 and acquisition of effective immune responses.21,22 It has been suggested that cellular immune mechanisms could protect individuals with thalassemia and sickle cell trait23,24 and that alterations of the RBC membrane lead to antibody and immune opsonisation reactions.21,25 Increased levels of immunoglobin G (IgG) with specificity for variant surface antigens (anti-VSA IgG) antibodies have been observed in HbS and HbC individuals,22,26,27 whereas other studies have failed to detect higher levels of malaria specific IgG antibodies in HbS or G6PD-deficient individuals.2833

The aim of our study was to examine whether individuals carrying sickle cell trait, alpha+-thalassemia, or G6PD deficiency have a reduced the risk of suffering from uncomplicated malaria and whether such protection could be explained by increased levels of anti-malarial IgG. In a 6-month longitudinal study, we collected morbidity data in a cohort of 0.5-to 19-year-old individuals living in a high-transmission area in northeastern Tanzania and measured the plasma levels of anti-malaria IgG against P. falciparum variant surface antigens (VSAs) and conserved glutamate-rich protein (GLURP) at the beginning and end of the study.

MATERIALS AND METHODS

Study site and populations.

As part of an immuno-epidemiologic survey of malaria, a 6-month longitudinal study was conducted between April and September 2001 in Mgome village in Tanga region, northeastern Tanzania. Malaria morbidity was assessed by active and passive case detection as described in detail elsewhere.34 The Mgome village is situated in an area with year-round intense transmission of P. falciparum. For this study, paired samples of plasma and filter paper blot spots were collected in April and September (before and after the peak malaria transmission season, respectively) from 202 children between 0.5 and 19 years of age. The point prevalence of P. falciparum parasitemia at the beginning of the study in April 2001 was 81% (N = 250) and varied only slightly during the study period. Parasite densities and levels of hemoglobin were as previously described.34 An episode of uncomplicated malaria was defined as a patient presenting with an axillary temperature ≥ 37.5°C and/or a history of fever within 48 hours and a positive blood slide with asexual parasites > 5,000/μL. Children were grouped as “no cases” if no malaria was detected and grouped as “cases” if they presented with one or more malaria episodes during the 6-month follow-up period. After an episode, the study participant was censored the following 4 weeks to ensure that the infection caused by this malaria episode was only recorded once. Written informed consent was obtained from all study participants or from his/her parent or guardian. Ethical approval was granted by the Medical Research Coordinating Committee, Dar Es Salaam, Tanzania.

DNA extraction and genotype screening.

DNA was extracted from filter papers in a 96-well plate format using Chelex-100 as described previously.35 Sickle cell trait and G6PD deficiency were determined by screening DNA for single nucleotide polymorphisms (SNPs) in the β-hemoglobin (A18T) and G6PD (G202A) genes by a simple high-throughput method using polymerase chain reaction (PCR), sequence-specific oligonucleotide probes (SSOPs), and ELISA-based technology.36 Individuals with no β-hemoglobin A18T mutation were HbAA, those heterozygous for the mutation were HbAS (sickle cell trait), and those homozygous for the mutation were classified HbSS (sickle cell disease). Individuals with no G202A mutation were classified G6PD B, those heterozygous for the G202A mutation (G6PD A/A− or B/A−) were classified G6PD A, and those homozygous or hemizygous (males) for the G202A mutation were classified G6PD A−, the G6PD deficiency phenotype. The prevalence of alpha+-thalassemia was determined by detection of the African α-globin deletion, α3.7, by PCR as described previously.37

Antibody assays.

The plasma IgG antibody level to GLURP (purified his-tag recombinant R0-GLURP) was measured by ELISA as reported in detail previously.38 The plasma level to VSAs expressed on erythrocytes of six in vitro–cultured parasite isolate was measured by flow cytometry as previously described.39 The parasites were obtained from randomly selected children living in Mgome village who carried an asymptomatic infection during the cross-sectional survey in April 2001. Each isolate had a unique genotype as determined by msp1 and msp2 PCR analysis. The geometric mean fluorescence index (MFI) was recorded as a measure of anti-VSA IgG reactivity with specificity for each particular parasite isolate. Non-specific labeling was evaluated by analysis of ethidium bromide–negative RBCs.

The cut-off for a positive anti-GLURP and anti-VSA IgG response, respectively, was defined as an antibody level above the mean level plus 2 SD of a group of negative control plasma samples from Danish donors never exposed to malaria. Because all children had anti-VSA antibodies against the tested isolates, the median of VSA IgG antibody levels were used to group children into “low” and “high: responders, respectively.

Statistical analysis.

All data were double-entered and analyzed statistically using Stata/SE version 8 and SigmaStat version 3.0. Differences in proportions were analyzed using the χ2 test or Fisher exact test. Associations between independent variables (RBC polymorphisms as binary variables) and dependent variables (presence of febrile episodes and means of hemoglobin levels, parasite densities, and antibody levels) were determined by logistic regression models, adjusting for the confounding effect of age and age2. Levels of parasite densities and antibodies were normalized by log transformation. Comparison of linear or log-transformed mean values between two or more groups were made with the Student unpaired t test and ANOVA test, respectively. The non-parametric Mann-Whitney rank sum test was applied when data were not normally distributed. P < 0.05 was considered statistically significant.

RESULTS

General characteristics of the children in April 2001 are summarized in Table 1. The data from the survey in September 2001 did not differ significantly with respect to variables studied or differences reported for other than the variables mentioned below.

Frequency of RBC polymorphisms.

Sickle cell trait (HbAS) was present in 14.9% of the children. No child was homozygous HbSS. The frequency of alpha+-thalassemia was 44.6% (heterozygous) and 9.7% (homozygous), resulting in a carriage rate (heterozygous + homozygous) of 54.3%. The frequency of G6PD deficiency was 10.7% (heterozygous) and 5.1% (homozygous), resulting in a carriage rate (heterozygous + homozygous) of 15.8%. All genotype polymorphisms were equally distributed among different ages and between males and females, with the exception of heterozygous G6PD (X-linked gene), which was not present in males (data not shown). Seven samples could not be amplified for assessment of alpha+-thalassemia, and six samples could not be amplified for assessment of the G6PD genotype.

Associations between RBC polymorphisms and parasitemia and anemia.

Regardless of age, the proportion of children with parasite infection in April 2001 was lower in children with sickle cell trait than in children without (χ2 = 5.32, P = 0.02), but this was not the case in September 2001 (χ2 = 0.83, P = 0.36). No differences in parasite prevalence rate were found with respect to alpha+-thalassemia or G6PD deficiency. Similarly, geometric mean parasite densities tended to be lower in children with HbAS compared with children with HbAA (P = 0.07), whereas associations between parasite densities and alpha+-thalassemia or G6PD deficiency were not found in April (Table 1) or September (data not shown).

Children homozygous for alpha+-thalassemia were at a higher risk of being mildly anemic (hemoglobin levels < 11 g/dL) in both April (OR = 0.27; 95% CI = 0.08–0.96; P = 0.04) and September (OR = 0.25; 95% CI = 0.08–0.84; P = 0.03, both adjusted for the confounding effect of age). This was not the case in children with sickle cell trait or G6PD deficiency.

Associations between RBC polymorphisms and febrile episodes.

During the 6-month study period, 176 of 202 children adequately completed the clinical follow-up. Among these, 56 (32%) had one or more febrile malaria episodes confirmed by malaria microscopy (parasites > 5,000/μL). The risk of developing a febrile malaria episode decreased with age (OR = 0.67; 95% CI = 0.58–0.79; P < 0.001). Logistic regression models adjusting for the confounding effect of age showed a reduced risk of febrile malaria episodes in children with alpha+-thalassemia. The decreased susceptibility in children with alpha+-thalassemia was significant both for heterozygous (OR = 0.30; 95% CI = 0.10–0.85; P = 0.02), homozygous (OR = 0.12; 95% CI = 0.02–0.83; P = 0.03), and homozygous and heterozygous combined (OR = 0.26; 95% CI = 0.09–0.71; P = 0.008; Table 2). The risk was mainly reduced in children older than 5 years of age (Table 3). No statistically significant reduction in malaria risk could be detected for any of the other RBC polymorphisms, and the conclusions did not change when the regression models also were adjusted for the presence of other genotypes (i.e., HbAS and G6PD deficiency).

Associations between RBC polymorphisms and antibodies.

We assessed the association between RBC polymorphisms and malaria antibodies by comparing children with or without RBC polymorphisms with respect to the presence and levels of plasma IgG antibodies against recombinant GLURP-R0 and VSA expressed by six heterologous parasite isolates.

No significant differences in levels of anti-GLURP IgG or anti-VSA IgG (antibody levels against the six parasite isolates tested independently and in total) were detected for any of the RBC polymorphisms in either April (Table 1) or September. The level of anti-VSA IgG (Figure 1A) and anti-GLURP IgG (Figure 1B) in April increased by age both in children with and without alpha+-thalassemia. A similar tendency was found for antibody levels in the September survey. Furthermore, in logistic regression models adjusting for age and parasite density in the April survey, no significant differences in the likelihood of having a measurable anti-GLURP IgG response could be detected between children with or without sickle cell trait (OR = 2.65; 95% CI = 0.51–13.8; P = 0.37), alpha+-thalassemia (OR = 0.65; 95% CI = 0.26–1.64; P = 0.24), or G6PD deficiency (OR = 1.09; 95% CI = 0.87–1.36; P = 0.45). Similarly, there was no statistically significant associations between anti-VSA IgG in children with or without the sickle cell trait (OR = 0.68; 95% CI = 0.01–3.39; P = 0.26), alpha+-thalassemia (OR = 0.70; 95% CI = 0.19–2.58; P = 0.59), or G6PD deficiency (OR = 1.06; 95% CI = 0.88–1.27; P = 0.54). The levels of anti-GLURP and anti-VSA IgG were not influenced by the density of parasites. Logistic regression models with data from the September survey gave similar results as the data obtained from the April survey.

DISCUSSION

In this study, we observed a reduced risk of uncomplicated malaria episodes in children homozygous and heterozygous for alpha+-thalassemia, and the protective effect of alpha+-thalassemia was most pronounced in children older than 5 years of age. In line with other studies, we found that alpha+-thalassemia was associated with lower hemoglobin levels but not lower parasite densities.14,15,40

The protective effect of alpha+-thalassemia against severe forms of malaria has been well described,8,9,41,42 whereas its role against uncomplicated disease is less clear. In Papua New Guinea, mild malaria incidences were not reduced in children with alpha+-thalassemia,42 whereas a study from Vanuatu reported that alpha+-thalassemia was associated with even higher incidences of uncomplicated malaria (mainly P. vivax) and that this was more pronounced in the youngest children.15 In line with our results, children with alpha+-thalassemia were associated with reduced risk against also uncomplicated malaria in studies from Kenya (although not significantly)14 and Nepal.41 The reasons for the discrepancy in results between the different studies could be because of differences in transmission intensity, case definitions, or age groups included.

The observed age-specific protective effect suggests that the reduced risk is mediated through an interaction between immune processes and the innate mechanisms of the genetic trait, which allows the immune system to control malaria earlier or more effectively in children with alpha+-thalassemia as suggested for individuals with hemoglobin S and C.26,43 This notion is not contradicted by a recent study suggesting that the protective effect of sickle cell trait against severe anemia and all-cause mortality mainly was found in children 2–16 months of age,7 because it is in this age group that children acquire immunity to these malaria syndromes. Our results also showed that children younger than 5 years of age with alpha+-thalassemia were almost as susceptible to uncomplicated malaria as children without alpha+-thalassemia. One could speculate that alpha+-thalassemia is an advantage against severe malaria predominantly in infants (< 2 years), whereas its protective effects against uncomplicated malaria would be more pronounced in older children (> 5 years) on improved immunity, as shown for the sickle cell trait.43 However, based on the small number of cases and the limited sample size, any conclusions based on the age-stratified analysis should be made with caution.

To study whether this apparent partial protection might be related to acquired humoral immunity, we measured antimalarial antibodies with specificities that have previously been associated with protective immunity.44,45 We hypothesized that the protective effector mechanisms against severe and uncomplicated malaria are functionally similar, and therefore, that modifications of RBC membranes in individuals with alpha+-thalassemia could imply an increase of antibody binding and consequently improvement of immune clearance against both severe and uncomplicated malaria. A similar mechanism has been proposed in RBCs expressing hemoglobin C.25 Several studies have identified high levels of antibodies to both GLURP44,46 and VSA against homologous and heterologous parasite isolates47,48 as predictors of reduced parasite density and malaria morbidity, and these targets were therefore chosen for analysis. However, in this study, we did not find any significant associations between increased levels or prevalence of IgG antibodies to VSA or GLURP and decreased malaria susceptibility in children with alpha+-thalassemia.

One explanation for this could be that antibody responses to other P. falciparum antigens than those measured are of importance, but it could also be that there are differences in the fine specificity or the functional capacity of the antibodies between the children with and without alpha+-thalassemia. Alternatively, alterations of the RBC membrane associated with RBC polymorphisms could enhance cellular immune mechanisms, and distinctive distributions of dendritic cells and monocytes associated with sickle cell trait and alpha+-thalassemia could contribute to protection.49

The prevalence of parasites were lower in children with sickle cell trait, supporting its protection against high parasitemia as shown by others.12,29 However, in contrast to previous findings,10,11,29,43 it was not associated with reduced risk of uncomplicated malaria in our study. We did not observe differences in GLURP and VSA antibody levels between children with and without HbAS. This contrasts previous observations showing that children with HbAS had higher levels of anti-VSA IgG,22,27 but is in line with other studies that also failed to show differences in malaria antibody levels between children with or without HbAS.2832 Recently Verra and others26 showed that carriers of HbS and HbC in a low-transmission urban area, but not in a rural high-transmission area, had higher immune responses to a variety of malaria antigens. They suggested that this could reflect a saturated immunity in individuals living under high malaria exposure, and this might also explain why we did not detect a difference in antibody prevalence in this study. The hypothesis that antibodies mediate protection in individuals with RBC polymorphisms was further challenged by Sarr and others,33 who observed lower levels of IgG antibodies in children with HbAS and G6PD deficiency to the merozoite antigens MSP2 and RESA. In our study, children with G6PD deficiency did not show a reduced risk of uncomplicated malaria or higher levels of malaria antibodies, which is in line with previous findings.4,33

There could be several reasons for not detecting differences in antibody response between the groups: 1) the limited sample size diminished the statistical power to detect an effect of the polymorphisms and their relation to significant differences in antibody responses; 2) antibodies against VSA on parasites causing severe malaria might correlate better with protection than antibodies against parasite isolates expressing VSAs characteristic for uncomplicated malaria; 3) considering short-term fluctuations in antibody levels, measuring antibody responses only twice, irrespective of the clinical status of the child at the time of blood sampling, might not adequately reflect the actual immune status of the child.

We showed that the frequency of alpha+-thalassemia in this study area is higher than the frequency of HbS and that it correlates strongly with malaria endemicity.50 A reason for not observing an advantageous effect of HbAS against uncomplicated malaria in this population could be because of the low frequency of the HbS allele, which did not allow for such studies. This low frequency could again be explained by the dominance of the alpha+-thalassemia polymorphism. Recently, it was predicted that, although alpha+-thalassemia and sickle cell trait independently were associated with resistance to clinical malaria, their protective effect was diminished when co-inherited.51

In conclusion, our findings suggest that alpha+-thalassemia is associated with protection against episodes of uncomplicated malaria, and this advantageous effect seems to be more predominant in children older than 5 years of age but seemed unrelated to antibody responses against VSAs and GLURP.

Table 1

Baseline characteristics of children with or without RBC polymorphisms, April 2001

Sickle cell traitAlpha+-thalassemiaG6PD deficiency
GenotypeHbAAHbASαα/αααα/α−α−/α−B/B, B/A, A/AB/A−, A/A−A−/A −
Seven samples could not be amplified for assessment for alpha+-thalassemia and six samples for assessment of G6PD genotype.
* Median (IQR).
† Geometric mean (95% CI).
‡ Mean (SD).
§ Proportion of children with VSA IgG antibody level above the median (high responders).
¶ Lower parasite densities, but not significantly, in children with HbAS compared with children with HbAA as calculated by the Student t test (P = 0.07).
** Significantly different from children with HbAA group as calculated by χ2.
Numbers, N (%)172 (85.1)30 (14.9)89 (45.6)87 (44.6)19 (9.7)165 (84.2)21 (10.7)10 (5.1)
Age, years*5.8 (4.9–6.6)6.7 (4.4–11.8)5.7 (4.7–6.6)6.0 (4.9–9.7)5.3 (3.2–9.7)5.9 (5.2–6.8)5.0 (3.5–10.9)3.6 (1.3–8.5)
Sex, males, %46.543.346.043.752.651.30.050.0
Parasites/μL†446 (316–631)209 (79–501)¶426 (267–708)338 (210–556)331 (126–795)393 (275–562)392 (118–1,318)912 (165–5,011)
Parasite positive, N (%)145 (84.3)20 (66.6)**73 (82.0)71 (81.6)15 (79.0)133 (80.7)17 (81.0.)9 (90.0)
Hemoglobin g/dL‡10.8 (1.8)11.6 (2.1)11.0 (2.0)10.9 (1.7)10.1 (1.7)11.0 (1.9)10.5 (1.9)10.6 (2.2)
VSA IgG †35.8 (27.9–44.0)36.5 (20.0–66.0)32.5 (20.9–43.6)38.9 (30.0–50.1)34.1 (17.5–66.1)38.0 (31.6–48.9)31.6 (16.2–61.6)33.9 (10.0–58.8)
VSA IgG > median, N (%)§84 (49.4)16 (53.3)43 (48.9)45 (52.3)10 (52.6)84 (51.2)8 (40.0)4 (80.0)
GLURP IgG†17.3 (14.0–21.4)13.5 (7.8–23.4)17.6 (12.5–24.5)17.2 (12.9–22.9)10.5 (5.9–18.6)17.9 (3.2–22.3)11.0 (6.3–13.8)11.2 (6.9–36.1)
GLURP IgG positive, N (%)103 (76.3)22 (73.3)55 (79.7)54 (78.3)12 (80.0)104 (77.5)11 (78.6)5 (75.0)
Table 2

Risk of febrile malaria episodes in children with or without RBC polymorphisms during the 6-month follow-up

GenotypeCases*/total N (%)Unadjusted OR (95% CI)PAdjusted OR (95% CI) †P
Of the 176 children completing follow-up, only 159 DNA samples were available for screening. Therefore, the total number of samples with both follow-up data and availability for screening was as follows: HbAS, 158 (1 missing amplification); alpha+-thalassemia, 152 (7 missing amplification); G6PD deficiency, 152 (7 missing amplification).
* A child with one or more episodes of uncomplicated febrile malaria during the 6-month follow-up period.
† Adjusted for age and age2.
‡ Heterozygous and homozygous alpha+-thalassemia combined.
HbAA47/135 (34.8)11
HbAS8/23 (34.8)1.00 (0.40–2.53)1.01.55 (0.51–4.77)0.44
αα/αα32/71 (45.1)11
αα/α−15/67 (22.4)0.35 (0.17–0.74)0.0060.30 (0.10–0.85)0.02
α−/α−3/14 (21.4)0.33 (0.09–1.29)0.110.12 (0.02–0.83)0.03
αα/α−, α−/α−‡18/81 (22.2)0.35 (0.17–0.70)0.0030.26 (0.09–0.71)0.008
G6PD (B)47/127 (37.0)11
G6PD (A)6/17 (35.3)0.93 (0.32–2.67)0.890.68 (0.17–2.77)0.59
G6PD (A−)3/8 (37.5)1.02 (0.23–4.46)0.980.24 (0.03–2.22)0.21
Table 3

Proportions of children with one or more episodes of uncomplicated malaria by age and alpha+-thalassemia status

Age (years)αα/αα*αα/α−, α−/α−*OR (95% CI)P
* Number of children with febrile episodes/all children (%) during the 6-month follow-up period.
† Differences in proportions of experiencing uncomplicated malaria between children with (αα/α−, α−/α−) and without (αα/αα) alpha+-thalassemia. OR adjusted for age, parasite density, and interaction between age and alpha+-thalassemia.
< 320/21 (95.2)14/17 (82.4)0.23 (0.02–2.50)0.23
3–54/12 (33.3)3/14 (21.4)0.55 (0.10–3.15)0.49
> 58/38 (21.0)1/50 (2.0)0.08 (0.01–0.64)0.01
Figure 1.
Figure 1.

Age-specific levels of malaria antibodies in children with or without alpha+-thalassemia versus without, adjusted for parasite density. A, Log-transformed arbitrary units of mean fluorescence intensities (MFIs) of accumulated IgG antibodies with specificity for variant surface antigens on six heterologous P. falciparum parasite isolates. B, Log-transformed arbitrary units of OD values of IgG antibody response with specificity to recombinant GLURP-R0 antigen. Data presented are from April 2001. Normal, αα/αα; alpha +-thalassemia, αα/α− or α−/α−. Age groups in years: 0–3, 3–5, and 5–19. Number of children in each group: see Table 3. Box plots show medians with 25th and 75th percentiles, whiskers for 10th and 90th percentiles, and dots for outliers. *Higher levels of anti-GLURP IgG, but not significantly, in children with alpha+-thalassemia 3–5 years of age as calculated by the Student unpaired t test.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 5; 10.4269/ajtmh.2008.78.714

*

Address correspondence to Anders Enevold, Centre for Medical Parasitology, Institute for International Health, Immunology and Microbiology, Øster Farimagsgade 5, Building 22+23, PO Box 2099, 1014 Copenhagen K, Denmark. E-mail: enevold@cmp.dk

Authors’ addresses: Anders Enevold, Michael Alifrangis, Ib C. Bygbjerg, and Thor G. Theander, Centre for Medical Parasitology, Institute for International Health, Immunology and Microbiology, CSS, Øster Farimagsgade 5, Building 22+23, PO Box 2099, 1014 Copenhagen K, Denmark, Tel: 45-3532-7680, Fax: 45-3532-7851, E-mails: enevold@cmp.dk, alifrangis@cmp.dk,I.Bygbjerg@pubhealth.ku.dk, and theander@cmp.dk. John P. Lusingu, Bruno Mmbando, and Martha M. Lemnge, National Institute for Medical Research, Tanga Medical Research Centre, PO Box 5004, Tanga, Tanzania, Tel: 255-2726-46084, Fax: 255-2726-43869, E-mails: jlusingu@tanga.mimcom.net, brmm@pubhealth.ku.dk, and mlemnge@tanga.mimcom.net. Lasse S. Vestergaard, Department of Epidemiology, Building 37/219, Statens Serum Institute, Copenhagen, Denmark, Tel: 45-32683-695, E-mail: lav@ssi.dk.

Acknowledgments: The authors thank all study participants including their parents/guardians, as well as village helpers and health management teams in Tanga region for participation. We highly appreciate the technical assistance of Maiken Visti, Juma Akida, and Hatibu Athumani. The study was conducted under the auspices of the Joint Malaria Programme, a collaborative research initiative between Centre for Medical Parasitology at the University of Copenhagen, Kilimanjaro Christian Medical College, London School of Hygiene and Tropical Medicine and the Tanzania National Institute for Medical Research.

Financial support: The field study was funded by ENRECA Programme Grant 104.Dan.8.L.312. AE was supported by DANIDA Grant 91203.

REFERENCES

  • 1

    Mcgregor IA, 1987. Malarial immunity—current trends and prospects. Ann Trop Med Parasitol 81 :647–656.

  • 2

    Allison AC, 1964. Polymorphism and natural selection in human populations. Cold Spring Harb Symp Quant Biol 29 :137–149.

  • 3

    Flint J, Hill AVS, Bowden DK, Oppenheimer SJ, Sill PR, Serjeantson SW, Banakoiri J, Bhatia K, Alpers MP, Boyce AJ, Weatherall DJ, Clegg JB, 1986. High-frequencies of alpha-thalassemia are the result of natural-selection by malaria. Nature 321 :744–750.

    • Search Google Scholar
    • Export Citation
  • 4

    Ruwende C, Khoo SC, Snow AW, Yates SNR, Kwiatkowski D, Gupta S, Warn P, Allsopp CEM, Gilbert SC, Peschu N, Newbold CI, Greenwood BM, Marsh K, Hill AVS, 1995. Natural-selection of hemizygotes and heterozygotes for G6PD deficiency in Africa by resistance to severe malaria. Nature 376 :246–249.

    • Search Google Scholar
    • Export Citation
  • 5

    Beutler E, 1991. Glucose-6-phosphate-dehydrogenase deficiency. N Engl J Med 324 :169–174.

  • 6

    Weatherall DJ, Clegg JB, 2001. Inherited haemoglobin disorders: an increasing global health problem. Bull WHO 79 :704–712.

  • 7

    Aidoo M, Terlouw DJ, Kolczak M, McElroy PD, ter Kuile FO, Kariuki S, Nahlen BL, Lal AA, Udhayakumar V, 2002. Protective effects of the sickle cell gene against malaria morbidity and mortality. Lancet 359 :1311–1312.

    • Search Google Scholar
    • Export Citation
  • 8

    Mockenhaupt FP, Ehrhardt S, Gellert S, Otchwemah RN, Dietz E, Anemana SD, Bienzle U, 2004. alpha(+)-thalassemia protects African children from severe malaria. Blood 104 :2003–2006.

    • Search Google Scholar
    • Export Citation
  • 9

    Williams TN, Wambua S, Uyoga S, Macharia A, Mwacharo JK, Newton CRJC, Maitland K, 2005. Both heterozygous and homozygous alpha(+) thalassemias protect against severe and fatal Plasmodium falciparum malaria on the coast of Kenya. Blood 106 :368–371.

    • Search Google Scholar
    • Export Citation
  • 10

    Allison AC, 1954. Protection afforded by sickle-cell trait against subtertian malarial infection. BMJ 1 :290–294.

  • 11

    Hill AVS, Allsopp CEM, Kwiatkowski D, Anstey NM, Twumasi P, Rowe PA, Bennett S, Brewster D, Mcmichael AJ, Greenwood BM, 1991. Common West African Hla antigens are associated with protection from severe malaria. Nature 352 :595–600.

    • Search Google Scholar
    • Export Citation
  • 12

    Williams TN, Mwangi TW, Wambua S, Alexander ND, Kortok M, Snow RW, Marsh K, 2005. Sickle cell trait and the risk of Plasmodium falciparum malaria and other childhood diseases. J Infect Dis 192 :178–186.

    • Search Google Scholar
    • Export Citation
  • 13

    Guindo A, Fairhurst RM, Doumbo O, Wellems TE, Diallo D, 2007. X-linked G6PD deficiency protects hemizygous males but not heterozygous females against severe malaria. PLoS Med 4 :e66.

    • Search Google Scholar
    • Export Citation
  • 14

    Wambua S, Mwangi TW, Kortok M, Uyoga S, Macharia AW, Mwacharo JK, Weatherall DJ, Snow RW, Marsh K, Williams TN, 2006. The effect of alpha+ thalassaemia on the incidence of malaria and other diseases in children living on the coast of Kenya. PLoS Med 3 :e158.

    • Search Google Scholar
    • Export Citation
  • 15

    Williams TN, Maitland K, Bennett S, Ganczakowski M, Peto TEA, Newbold CI, Bowden DK, Weatherall DJ, Clegg JB, 1996. High incidence of malaria in alpha-thalassaemic children. Nature 383 :522–525.

    • Search Google Scholar
    • Export Citation
  • 16

    Oppenheimer SJ, Hill AVS, Gibson FD, Macfarlane SB, Moody JB, Pringle J, 1987. The interaction of alpha-Thalassemia with malaria. Trans R Soc Trop Med Hyg 81 :322–326.

    • Search Google Scholar
    • Export Citation
  • 17

    Roth EF, Raventossuarez C, Rinaldi A, Nagel RL, 1983. Glucose-6-phosphate-dehydrogenase deficiency inhibits in vitro growth of Plasmodium falciparum.Proc Natl Acad Sci USA 80 :298–299.

    • Search Google Scholar
    • Export Citation
  • 18

    Pasvol G, Weatherall DJ, Wilson RJM, 1978. Cellular mechanism for protective effect of haemoglobin-S against P. falciparum malaria. Nature 274 :701–703.

    • Search Google Scholar
    • Export Citation
  • 19

    Cappadoro M, Giribaldi G, O’Brien E, Turrini F, Mannu F, Ulliers D, Simula G, Luzzatto L, Arese P, 1998. Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum may explain malaria protection in G6PD deficiency. Blood 92 :2527–2534.

    • Search Google Scholar
    • Export Citation
  • 20

    Ayi K, Turrini F, Piga A, Arese P, 2004. 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 104 :3364–3371.

    • Search Google Scholar
    • Export Citation
  • 21

    Luzzi GA, Merry AH, Newbold CI, Marsh K, Pasvol G, 1991. Protection by alpha-thalassemia against Plasmodium falciparum malaria—modified surface-antigen expression rather than impaired growth or cytoadherence. Immunol Lett 30 :233–240.

    • Search Google Scholar
    • Export Citation
  • 22

    Marsh K, Otoo L, Hayes RJ, Carson DC, Greenwood BM, 1989. Antibodies to blood stage antigens of Plasmodium falciparum in rural Gambians and their relation to protection against infection. Trans R Soc Trop Med Hyg 83 :293–303.

    • Search Google Scholar
    • Export Citation
  • 23

    Pasvol G, 1992. A cellular mechanism for the protection by thalassemia against Plasmodium falciparum malaria. Br J Haematol 82 :267.

  • 24

    Bayoumi RA, Abuzeid YA, Abdulhadi NH, Saeed BO, Theander TG, Hviid L, Ghalib HW, Nugud AHD, Jepsen S, Jensen JB, 1990. Cell-mediated immune-responses to Plasmodium falciparum purified soluble antigens in sickle-cell trait subjects. Immunol Lett 25 :243–250.

    • Search Google Scholar
    • Export Citation
  • 25

    Arie T, Fairhurst RM, Brittain NJ, Wellems TE, Dvorak JA, 2005. Hemogloblin C modulates the surface topography of Plasmodium falciparum-infected erythrocytes. J Struct Bio 150 :163–169.

    • Search Google Scholar
    • Export Citation
  • 26

    Verra F, Simpore J, Warimwe GM, Tetteh KK, Howard TOF, Bancone G, Avellino P, Blot I, Fegan G, Bull PC, Williams TN, Conway DJ, Marsh K, Modiano D, 2007. Haemoglobin C and S role in acquired immunity against Plasmodium falciparum malaria. PloS One e978.

    • Search Google Scholar
    • Export Citation
  • 27

    Cabrera G, Cot M, Migot-Nabias F, Kremsner PG, Deloron P, Luty AJF, 2005. The sickle cell trait is associated with enhanced immunoglobulin G antibody responses to Plasmodium falciparum variant surface antigens. J Infect Dis 191 :1631–1638.

    • Search Google Scholar
    • Export Citation
  • 28

    Jakobsen PH, Riley EM, Allen SJ, Larsen SO, Bennett S, Jepsen S, Greenwood BM, 1991. Differential antibody-response of Gambian donors to soluble Plasmodium falciparum antigens. Trans R Soc Trop Med Hyg 85 :26–32.

    • Search Google Scholar
    • Export Citation
  • 29

    Allen SJ, Bennett S, Riley EM, Rowe PA, Jakobsen PH, Odonnell A, Greenwood BM, 1992. Morbidity from malaria and immune-responses to defined Plasmodium falciparum antigens in children with sickle-cell trait in the Gambia. Trans R Soc Trop Med Hyg 86 :494–498.

    • Search Google Scholar
    • Export Citation
  • 30

    Le Hesran JY, Personne I, Personne P, Fievet N, Dubois B, Beyeme M, Boudin C, Cot M, Deloron P, 1999. Longitudinal study of Plasmodium falciparum infection and immune responses in infants with or without the sickle cell trait. Int J Epidemiol 28 :793–798.

    • Search Google Scholar
    • Export Citation
  • 31

    Maya DWM, Mavoungou E, Deloron P, Theisen M, Ntoumi F, 2006. Distribution of IgG subclass antibodies specific for Plasmodium falciparum glutamate-rich-protein molecule in sickle cell trait children with asymptomatic infections. Exp Parasitol 112 :92–98.

    • Search Google Scholar
    • Export Citation
  • 32

    Ntoumi F, Ekala MT, Makuwa M, Lekoulou F, Mercereau-Puijalon O, Deloron P, 2002. Sickle cell trait carriage: imbalanced distribution of IgG subclass antibodies reactive to Plasmodium falciparum family-specific MSP2 peptides in serum samples from Gabonese children. Immunol Lett 84 :9–16.

    • Search Google Scholar
    • Export Citation
  • 33

    Sarr JB, Pelleau S, Toly C, Guitard J, Konate L, Deloron P, Garcia A, Migot-Nabias F, 2006. Impact of red blood cell polymorphisms on the antibody response to Plasmodium falciparum in Senegal. Microbes Infect 8 :1260–1268.

    • Search Google Scholar
    • Export Citation
  • 34

    Lusingu JPA, Vestergaard LS, Mmbando BP, Drakeley CJ, Jones C, Akida J, Savaeli ZX, Kitua AY, Lemnge MM, Theander TG, 2004. Malaria morbidity and immunity among residents of villages with different Plasmodium falciparum transmission intensity in North-Eastern Tanzania. Malar J 3 :26.

    • Search Google Scholar
    • Export Citation
  • 35

    Pearce RJ, Drakeley C, Chandramohan D, Mosha F, Roper C, 2003. Molecular determination of point mutation haplotypes in the dihydrofolate reductase and dihydropteroate synthase of Plasmodium falciparum in three districts of northern Tanzania. Antimicrob Agents Chemo 47 :1347–1354.

    • Search Google Scholar
    • Export Citation
  • 36

    Enevold A, Vestergaard LS, Lusingu J, Drakeley CJ, Lemnge MM, Theander TG, Bygbjerg IC, Alifrangis M, 2005. Rapid screening for glucose-6-phosphate dehydrogenase deficiency and haemoglobin polymorphisms in Africa by a simple high-throughput SSOP-ELISA method. Malar J 4 :61.

    • Search Google Scholar
    • Export Citation
  • 37

    Liu YT, Old JM, Miles K, Fisher CA, Weatherall DJ, Clegg JB, 2000. Rapid detection of alpha-thalassaemia deletions and alpha-globin gene triplication by multiplex polymerase chain reactions. Br J Haematol 108 :295–299.

    • Search Google Scholar
    • Export Citation
  • 38

    Lusingu JPA, Vestergaard LS, Alifrangis M, Mmbando BP, Theisen M, Kitua AY, Lemnge MM, Theander TG, 2005. Cytophilic antibodies to Plasmodium falciparum glutamate rich protein are associated with malaria protection in an area of holoendemic transmission. Malar J 4 :48.

    • Search Google Scholar
    • Export Citation
  • 39

    Staalsoe T, Giha HA, Dodoo D, Theander TG, Hviid L, 1999. Detection of antibodies to variant antigens on Plasmodium falciparum- infected erythrocytes by flow cytometry. Cytometry 35 :329–336.

    • Search Google Scholar
    • Export Citation
  • 40

    Mockenhaupt FP, Falusi AG, May J, Ademowo OG, Olumese PE, Meyer CG, Bienzle U, 1999. The contribution of alpha(+)-thalassaemia to anaemia in a Nigerian population exposed to intense malaria transmission. Trop Med Int Health 4 :302–307.

    • Search Google Scholar
    • Export Citation
  • 41

    Modiano G, Morpurgo G, Terrenato L, Novelletto A, Dirienzo A, Colombo B, Purpura M, Mariani M, Santachiarabenerecetti S, Brega A, Dixit KA, Shrestha SL, Lania A, Wanachiwanawin W, Luzzatto L, 1991. Protection against malaria morbidity—near-fixation of the alpha-thalassemia gene in a Nepalese population. Am J Hum Genet 48 :390–397.

    • Search Google Scholar
    • Export Citation
  • 42

    Allen SJ, O’Donnell A, Alexander NDE, Alpers MP, Peto TEA, Clegg JB, Weatherall DJ, 1997. Alpha(+)-thalassemia protects children against disease caused by other infections as well as malaria. Proc Natl Acad Sci USA 94 :14736–14741.

    • Search Google Scholar
    • Export Citation
  • 43

    Williams TN, Mwangi TW, Roberts DJ, Alexander ND, Weatherall DJ, Wambua S, Kortok M, Snow RW, Marsh K, 2005. An immune basis for malaria protection by the sickle cell trait. PLoS Med 2 :441–445.

    • Search Google Scholar
    • Export Citation
  • 44

    Dodoo D, Theisen M, Kurtzhals JAL, Akanmori BD, Koram KA, Jepsen S, Nkrumah FK, Theander TG, Hviid L, 2000. Naturally acquired antibodies to the glutamate-rich protein are associated with protection against Plasmodium falciparum malaria. J Infect Dis 181 :1202–1205.

    • Search Google Scholar
    • Export Citation
  • 45

    Bull PC, Lowe BS, Kortok M, Molyneux CS, Newbold CI, Marsh K, 1998. Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria. Nat Med 4 :358–360.

    • Search Google Scholar
    • Export Citation
  • 46

    Oeuvray C, Theisen M, Rogier C, Trape JF, Jepsen S, Druilhe P, 2000. Cytophilic immunoglobulin responses to Plasmodium falciparum glutamate-rich protein are correlated with protection against clinical malaria in Dielmo, Senegal. Infect Immun 68 :2617–2620.

    • Search Google Scholar
    • Export Citation
  • 47

    Marsh K, Howard RJ, 1986. Antigens induced on erythrocytes by P. falciparum: expression of diverse and conserved determinants. Science 231 :150–153.

    • Search Google Scholar
    • Export Citation
  • 48

    Bull PC, Marsh K, 2002. The role of antibodies to Plasmodium falciparum-infected-erythrocyte surface antigens in naturally acquired immunity to malaria. Trends Microbiol 10 :55–58.

    • Search Google Scholar
    • Export Citation
  • 49

    Urban BC, Shafi MJ, Cordery DV, Macharia A, Lowe B, Marsh K, Williams TN, 2006. Frequencies of peripheral blood myeloid cells in healthy Kenyan children with alpha+ thalassemia and the sickle cell trait. Am J Trop Med Hyg 74 :578–584.

    • Search Google Scholar
    • Export Citation
  • 50

    Enevold A, Alifrangis M, Sanchez J, Carneiro I, Roper C, Børsting C, Lusingu J, Vestergaard L, Lemnge M, Morling N, Riley E, Drakeley C, 2007. Associations between alpha +-Thalassemia and Plasmodium falciparum Malarial Infection in Northeastern Tanzania. J Infect Dis 196 :451–459.

    • Search Google Scholar
    • Export Citation
  • 51

    Williams TN, Mwangi TW, Wambua S, Peto TEA, Weatherall DJ, Gupta S, Recker M, Penman BS, Uyoga S, Macharia A, Mwacharo JK, Snow RW, Marsh K, 2005. Negative epistasis between the malaria-protective effects of alpha(+)-thalassemia and the sickle cell trait. Nat Gen 37 :1253–1257.

    • Search Google Scholar
    • Export Citation
Save