Increased Microerythrocyte Count in Homozygous α+-Thalassaemia Contributes to Protection against Severe Malarial Anaemia

et al. (2008) Increased microerythrocyte count in homozygous a- thalassaemia contributes to protection against severe malarial anaemia. PLoS Med 5(3): e56. doi:10. 1371/journal.pmed.0050056 Increased Microerythrocyte Count in Homozygous a-Thalassaemia Contributes to Protection against Severe Malarial Anaemia Freya J. I. Fowkes 0 1 2 3 Stephen J. Allen 0 1 2 3 Angela Allen 0 1 2 3 Michael P. Alpers 0 1 2 3 David J. Weatherall 0 1 2 3 Karen P. Day 0 1 2 3 0 a Current address: School of Medicine, Swansea University , Swansea , United Kingdom 1 Academic Editor: Geoffrey Pasvol, Imperial College London , United Kingdom 2 1 Peter Medawar Building for Pathogen Research and Department of Zoology, University of Oxford , Oxford , United Kingdom , 2 Department of Medical Parasitology, New York University School of Medicine , New York , New York, United States of America, 3 The Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital , Oxford , United Kingdom , 4 PNG Institute of Medical Research , Goroka , Papua New Guinea 3 b Current address: Centre for International Health, Curtin University of Technology , Perth , Australia - The increased erythrocyte count and microcytosis in children homozygous for a-thalassaemia may contribute substantially to their protection against SMA. A lower concentration of Hb per erythrocyte and a larger population of erythrocytes may be a biologically advantageous strategy against the significant reduction in erythrocyte count that occurs during acute infection with the malaria parasite Plasmodium falciparum. This haematological profile may reduce the risk of anaemia by other Plasmodium species, as well as other causes of anaemia. Other host polymorphisms that induce an increased erythrocyte count and microcytosis may confer a similar advantage. The Editors Summary of this article follows the references. The heritable haemoglobinopathy a-thalassaemia is one of the most common monogenic disorders of humans [1]. The different forms of a-thalassaemia result from deletions (3.7 or 4.2 kb) or point mutations in one of the duplicated a-globin genes (aa/aa) on Chromosome 16 [1]. The heterozygous ( a/aa) and homozygous ( a/ a) states for a-thalassaemia are characterised by lower haemoglobin (Hb) concentration, mean cell volume (MCV) and mean cell Hb (MCH), but increased erythrocyte count compared with normal individuals [13]. This mild hypochromic, microcytic anaemia is more pronounced in individuals homozygous for a-thalassaemia compared to heterozygous individuals [13]. Haldanes proposal that the high frequencies of thalassaemias in malaria endemic regions were due to natural selection by malaria [4] is consistent with the strong geographical correlation between the frequency of a-thalassaemia and malaria in the Pacific region [5]. It has been suggested that a-thalassaemia protects by a direct interaction between the parasite (Plasmodium falciparum) and the altered thalassaemic erythrocyte, resulting in reduced parasite load [6]. However, in vitro experiments have failed to consistently demonstrate either a reduced ability of the malaria parasite to grow in and/or invade thalassaemic erythrocytes [717], and epidemiological studies have failed to demonstrate any consistent effect of a-thalassaemia against P. falciparum density [1822]. These findings argue against an altered physical interaction between the erythrocyte and the parasite. Case-control studies in Papua New Guinea (PNG) [18] and Africa [19,20,22,23] have demonstrated the protective effect of a-thalassaemia against severe malaria. Most studies show that a-thalassaemia homozygotes have considerable protection against SMA compared to heterozygotes [18,20,22,23], although one study showed equal odds of heterozygotes and homozygotes developing SMA [19]. We hypothesised that this striking protection against SMA may be due to the microcytosis and increased erythrocyte count in children homozygous for a-thalassaemia. We have reported previously a case-control study of children living in the north coastal region of PNG that showed that the odds ratio for SMA was 0.34 (95% confidence interval [CI] 0.160.73) in homozygous a-thalassaemia compared to normal individuals [15]. In the present study, we explore the consequences of a range of reductions of erythrocyte count on genotype-specific Hb levels by modelling the observed haematological data from the original case-control study. Materials and Methods Study Site The north coast of PNG provides a unique site to investigate the protective effect of a-thalassaemia against malaria. The frequency of a-thalassaemia is very high (68%) [1], and other host erythrocyte polymorphisms such as Southeast Asian ovalocytosis and glucose-6-phosphate dehydrogenase deficiency are relatively uncommon in this population (,7%) [18,21]. Importantly, sickle cell trait, a haemoglobinopathy proposed to be a serious confounder in epidemiological studies of a-thalassaemia in Africa [24,25], is absent in PNG. Data Collection In the case-control study, children with acute malaria were recruited from outpatient clinics and Madang General Hospital between October 1993 and February 1996 [18]. In children admitted to hospital, malaria was defined as a febrile illness with any degree of P. falciparum parasitaemia (as they had often received antimalarial treatment before admission), but without an alternative cause of illness identified during detailed clinical and laboratory investigation. Some of these children developed one or more severe manifestations of malaria, defined according to World Health Organization criteria [26,27]. For each case of severe malaria, a control child living in the community was selected randomly and individually matched to the index case for age, sex, ethnicity, season, and village. Children in the community frequently harbour chronic, asymptomatic P. falciparum, P. vivax, P. malariae, and P. ovale [28]. Analysis of the protective effect of a-thalassaemia against severe manifestations of malaria accounted for the matched pair design. Children attending clinics with P. falciparum parasitaemia 10,000/ll and no clinical or laboratory features of severe malaria or an alternative cause of an acute febrile illness were also recruited. To investigate the effect of the haematological characteristics according to a-globin genotype on anaemia associated with malaria infection of varying severity, we have pooled children from our original clinic and hospital malaria groups together. Venous blood was collected from all children at presentation. Thick and thin blood films were prepared, stained with Giemsa, and examined by microscopy for the presence of malaria parasites. Blood collected into EDTA was used for measurement of Hb, erythrocyte count, MCV, and MCH (Coulter MD8 instrument, Coulter Electronics). These measurements were done promptly after sample collection, and reliability of results was ensured by participation in (...truncated)