Protection against malaria in the hemoglobinopathies

INTRODUCTION — The history of genetics and the study of malaria are inextricably linked. The burden of disease due to malaria across much of the world has selected for a series of very visible traits of major medical importance, including the alleles of genes encoding hemoglobin, red cell enzymes, and membrane proteins. Furthermore, as might be expected from the intricate life cycle of the parasite in the human host, it now appears that many other genes may also influence the outcome of infection, including some that modulate the immune responses and others that encode for endothelial proteins.

The genetic resistance to malarial infection, particularly falciparum malaria, associated with the hemoglobinopathies, will be reviewed here. Resistance associated with abnormalities in red cell surface antigens or cytoskeleton is discussed separately. (See "Protection against malaria by abnormalities in red cell surface antigens and cytoskeletal proteins".)

REVIEW OF MALARIAL INFECTION — Plasmodium falciparum malaria, the deadly form of malaria, has a life cycle that includes alternating hosts: a sexual cycle in the insect vector (a female) Anopheles mosquito and a human cycle that includes a liver stage and an erythrocytic stage. Genetic resistance to the blood stage has been extensively documented, but the association of HLA class I allotypes with protection from malaria suggests genetic traits conferring resistance also exist during the hepatic stage of infection [1,2].

Genetic resistance to P. falciparum malaria at the erythrocytic stage involve one or more of the following mechanisms:

●Inhibition of merozoite entry into the red cell [3]

●Impairment in intracellular growth of the parasite

●Prevention of the erythrocyte lysis that occurs at the end of parasite maturation, which leads to release of merozoites into the bloodstream

●Enhanced phagocytosis of parasite-infected red cells [4-7]

●Reduced cytoadherence of infected erythrocytes to endothelial cells, uninfected red blood cells, platelets or antigen-presenting cells

●Enhanced immune responses to malarial infection

Genetic resistance at the erythrocytic stage has led to the selection of red cell genetic defects at gene frequencies (greater than 1 percent) that imply positive selective pressure [8,9]. These defects include hemoglobinopathies (eg, hemoglobins [Hbs] S, C, E, and thalassemia), erythrocyte membrane defects (eg, Southeast Asian ovalocytosis and some forms of elliptocytosis), Dantu blood group, glycophorins, metabolic abnormalities (eg, glucose-6-phosphate dehydrogenase [G6PD] deficiency), and other abnormalities of red blood cell function [10].

Although all of the four common types of human malaria (P. falciparum, ovale, vivax, and malariae) can theoretically cause selective pressure on the human population, and although P. vivax does cause severe disease and may also indirectly contribute to morbidity and mortality, this effect is most obvious with P. falciparum infection [9,11]. (See "Pathogenesis of malaria", section on 'Genetic factors'.)

MALARIA PROTECTION AND INHERITED FACTORS — The first observations suggesting that malaria is influenced by inherited factors were made in the study of malaria in North America in 1887, where Klebs and Tomassi commented on the apparent resistance to malaria of those of African descent, who "better than any other resist (malaria's) action."

However, it was AE Garrod who stated a clear hypothesis that constitutional factors may underlie the striking individual variation in the outcome of infectious disease.

Later, support for a contribution of genetic factors to malaria susceptibility came from malaria therapy, when patients with neurosyphilis were deliberately infected with malaria for treatment purposes; the marked variation in susceptibility observed in non-immune adults was accepted as general evidence for a genetic contribution.

Population genetic studies associated the prevalence of sickle hemoglobin (Hb S), thalassemias, and a range of red cell membrane and enzyme abnormalities, with regions where malaria was endemic. It has now been confirmed that many of these traits provide substantial protection against the disease.

Intriguingly, most of these red cell genetic defects in human populations have been selected during the last 3000 to 5000 years with the emergence of agriculture [12-14]. However, other factors also affect the outcome of individual malaria infections including previous exposure and immune status and determinants of parasite virulence.

Mackinnon and colleagues examined pedigree data collected during longitudinal cohort studies in Sri Lanka [15] and calculated 20 percent of the variation in the intensity of disease was explained by repeatable differences between patients, and approximately half of this variation was attributable to host genetics. A similar analysis of the total genetic contribution to malaria infection and disease in Africa showed 25 percent of the total variation in the incidence of hospital admission for malaria was explained by additively acting host genes. More recent estimates of the role of genetic traits in resistance to malaria suggests approximately one-third of the variability in the risk of severe and complicated malaria is explained by additive host genetic effects [16]. Interestingly, only 2 percent of this variation was due to the sickle cell trait, suggesting that the additive effect of the many other, largely undefined, genetic resistance traits is much greater than those at the globin loci [17].

A series of case-control and some cohort studies have explored the role of specific hemoglobinopathies in resistance to malaria. A meta-analysis of these case-control studies showed a decreased risk of severe P. falciparum malaria in individuals with Hb AS (odds ratio [OR] 0.09, 95% CI 0.06-0.12), Hb CC (OR 0.27, 95% CI 0.11-0.63), Hb AC (OR 0.83, 95% CI 0.67-0.96), homozygous alpha thalassemia (OR 0.63, 95% CI 0.48-0.83), and heterozygous alpha thalassemia (OR 0.83, 95% CI 0.74-0.92). However, only Hb AS showed protection from uncomplicated malaria (incidence rate ratio 0.69, 95% CI 0.61-0.79), while no hemoglobinopathies led to consistent protection from asymptomatic parasitemia [18].

FALCIPARUM MALARIA AND HEMOGLOBIN S — Early observations suggested that the peculiar distribution of sickle cell trait in African populations might result from a selective advantage against malaria [19,20]. However, the "malaria hypothesis" is generally ascribed to Haldane who, reflecting on the evolving descriptions of the global distribution of beta thalassemia, wrote in 1949:

"The corpuscles of the anaemic heterozygotes are smaller than normal, and more resistant to hypotonic solutions. It is at least conceivable that they are also more resistant to attacks by the sporozoa which cause malaria, a disease prevalent in Italy, Sicily, and Greece, where the gene is frequent" [21].

Since Haldane's first statement of his "malaria hypothesis," evidence that many genes provide protection from P. falciparum malaria has accumulated relentlessly. However, transmission experiments have suggested that the "protective hemoglobin variants" (eg, Hb S and Hb C) may be associated with an increase in parasite transmission from the human host to the Anopheles mosquito vector [22].

Sickle cell trait

Epidemiological evidence for protection — Hemoglobin (Hb) S is a variant form of hemoglobin due to an abnormal beta globin chain. Homozygotes for this mutation (Hb SS) suffer from sickle cell disease, a debilitating form of anemia associated with premature death in many areas where malaria is endemic. Nevertheless, it became increasingly obvious that the carrier state (sickle cell trait, Hb AS) was extremely common in much of sub-Saharan Africa. Support for this hypothesis of a balanced polymorphism was shown through the observation that sickle cell heterozygotes (Hb AS) were less likely to have parasites in their blood and were less likely to die from severe malaria than individuals with Hb AA [23]. (See "Sickle cell trait".)

Case-control studies have since confirmed that sickle cell trait is 90 percent protective against severe and complicated malaria (cerebral malaria and severe anemia) and 60 percent protective against clinical malaria leading to hospital admission [1,24]. A cohort study has shown that sickle trait confers 60 percent protection against mortality between 2 and 16 months of age in an area of high transmission [11]. Although there is some evidence for protection against mild clinical malaria [25-27], this is generally manifest as reduced parasite densities rather than protection against parasitemia per se.

Malaria infection strongly predisposes to bacteremia, often with non-Salmonella typhi species. In malaria endemic areas, Hb AS may protect not only from malaria but also from bacteremia (odds ratio [OR] 0.36; 95% CI 0.2-0.65) [28]. Those with Hb AS are also protected from neonatal conditions (perhaps indirectly) (OR 0.79; 95% CI 0.67-0.93) and malnutrition (OR 0.67; 95% CI 0.55-0.83) [29].

Hb AS may also confer protection from iron deficiency anemia. Using Mendelian randomization, Hb AS was shown to be associated with a 30 percent reduction in iron deficiency among children living in malaria-endemic countries in Africa, but not among individuals living in malaria-free areas [30]. Genetically predicted malaria risk was associated with an odds ratio of 2.65 for iron deficiency per unit increase in the log incidence rate of malaria. Incidentally, this study shows that an intervention that halves the risk of malaria episodes would also reduce the prevalence of iron deficiency in children in Africa by half.

The most comprehensive longitudinal studies of the role of sickle cell trait in protection from malaria have shown protection from mild disease and from severe disease but negative epistasis (or interaction) between the malaria-protective effects of alpha thalassemia and sickle cell trait. The protection afforded by each condition inherited alone was lost when the two conditions were inherited together, to such a degree that the incidence of both uncomplicated and severe P. falciparum malaria was close to baseline in children with both Hb AS and homozygosity for alpha⁺ thalassemia [31].

However, clinical epidemiology has yielded few clues regarding the mechanisms involved in this protection. An early report showed reduced parasite prevalence in heterozygotes (Hb AS) [23], and while subsequent studies did not always confirm these findings, a quantitative trait locus analysis of parasite density has shown that Hb S gene carriage protects individuals with severe P. falciparum malaria against hyperparasitemia [32].

Interestingly, in spite of this robust evidence for protection against clinical malaria in childhood, it may be that Hb AS does not protect against placental malaria across a range of parasitologic, clinical, and histologic outcomes [33].

Mechanisms — The mechanisms by which sickle cell trait protects against P. falciparum are not fully understood, and may be both innate and acquired [34,35]. However, two principal factors are increased sickling and impaired growth of the parasite during vascular sequestration.

●Increased sickling – Studies using cultured parasitized sickle cell trait red cells at physiologic levels of deoxygenation showed that cells infected with ring forms had accelerated sickling compared with noninfected cells [36,37]. Accelerated sickling might be beneficial by promoting removal of infected cells from the circulation, a phenomenon that has been called "suicidal" infection [37]. However, morphologic sickling did not occur in sickle cell trait cells containing trophozoites (which do not circulate since they are sequestered in the microcirculation), although abundant Hb S polymer was detectable by electron microscopy [36].

●Impaired parasite growth – P. falciparum grows normally in infected sickle cell trait cells exposed to 17 percent oxygen, but reducing the oxygen content to 3 percent results in parasite death within a few days [38]. This deoxygenation effect is more pronounced in Hb SC and Hb SS erythrocytes.

The reduced growth of malaria parasites in sickle cell trait cells compared with Hb A red cells after transfer from high to low oxygen conditions was shown to be due to formation of sickle hemoglobin [39]. This effect may be more biologically significant in reducing morbidity and mortality from malaria than a reduction in cytoadherence of infected red cells to the endothelium.

Even if infected sickle trait cells do not undergo morphologic sickling, the parasites are still hampered in the early trophozoite stage. P. falciparum growth is impaired in deoxygenated sickle trait cells [40-42], although the rate of parasite invasion appears to be intact [42]. Reduction in growth has been ascribed to dysregulated microRNA (miRNA) in Hb AS or Hb SS erythrocytes. A subset of erythrocyte miRNAs translocates into the parasite and reduces the growth of the parasites by integration of miR-451 and let-7i into essential parasite messenger RNAs (mRNAs) and so inhibition of translation [43].

Ultrastructural studies of parasitized sickle cell anemia cells have shown that, after six hours of deoxygenation, there is disruption of the parasitophorous vacuole and other membranes [44]. This membrane disruption in sickle cell trait erythrocytes may be due to puncture by spear-like polymers, which might account for the cell lysis observed with parasitized erythrocytes in sickle cell anemia. Another possibility is that the Hb S polymer, with its considerably right shifted oxygen equilibrium curve, kills the parasite by oxygen toxicity as it releases oxygen during the process of polymerization.

Several mechanisms, not mutually exclusive, have been proposed to explain impaired parasite growth or death in deoxygenated sickle trait erythrocytes or removal of parasites growing in sickle trait red cells from the circulation [43,45-53].

●Loss of potassium from red cells – The loss of potassium that accompanies sickling could be detrimental to parasite growth. Sickle erythrocytes have two abnormal mechanisms that facilitate potassium loss. One is the increased activity of the K-Cl cotransporter, due in part to absence of the normal inhibition of the transporter by low oxygen tension, along with retention of its ability to be activated by low pH; both of which occur at sites of stagnant circulation. The second mechanism is increased activity of the calcium-dependent (Gardos) potassium channel, due to a transient increase in cell calcium during sickling [54-56]. (See "Control of red blood cell hydration" and "Pathophysiology of sickle cell disease", section on 'Effects on the RBC'.)

●Loss of water from red cells – The osmotic loss of water that follows the loss of potassium increases the mean cell Hb S concentration and Hb S polymerization. (See "Pathophysiology of sickle cell disease", section on 'Effects on the RBC'.)

Parasite growth is normal in red cells that have a low potassium concentration induced by incubation with ouabain, which inhibits the Na-K-ATPase pump [57,58]. This observation suggests that water loss with promotion of polymerization of Hb S, not a low potassium content, is of primary importance for impairment in parasite growth.

●Inhibition of parasite growth by red cell miRNA – Erythrocyte microRNAs (miRNAs) are translocated into the parasite in a sequence-specific manner. In experimental systems, transfection of the erythrocyte miRNAs miR-451 and let-7i, which are enhanced in erythrocytes containing Hb AS, inhibit parasite growth. In these transfected red blood cells, erythrocyte miRNAs form chimeric transcripts with the 5' end of parasite mRNAs and inhibit translation of targeted parasite mRNAs, although these observations have not been supported by further evidence from animal or human infections [43].

●Increased phagocytosis – Enhanced phagocytosis of ring or the early intraerythrocytic form of the parasite may be an explanation for protection in glucose-6-phosphate dehydrogenase (G6PD) deficiency [46]. Malaria-infected erythrocytes containing Hb AS are also susceptible to enhanced phagocytosis of ring forms of the parasite [47].

Membrane-bound hemichromes, autologous immunoglobulin G (IgG) and complement C3c fragments, aggregated band 3, and phagocytosis by human monocytes were greater in rings developing in Hb AS erythrocytes than in controls, and phagocytosis of ring-parasitized Hb AS erythrocytes was predominantly complement mediated. Similar findings were reported for red cells in beta thalassemia trait and Hb H disease, but not for alpha thalassemia trait erythrocytes [6].

However, other groups measuring retention of malaria-infected red blood cells by microspheres in vitro or human spleens ex vivo have not shown that ring-stage parasites in Hb AS red cells are retained more than ring-stage parasites in Hb AA red cells [47].

●Altered remodeling of red cell cytoskeleton and trafficking of parasite proteins – There is also substantial evidence for altered actin polymerization in malaria-infected Hb AS cells. Hbs S and C affect the trafficking system that directs parasite-encoded proteins to the surface of infected erythrocytes. Hemoglobin oxidation may inhibit actin polymerization and disrupt the actin cytoskeleton and Maurer's clefts [48]. Maurer's clefts moved significantly faster in infected erythrocytes containing the Hb variants S and C than in infected wild-type erythrocytes due to an impaired actin network in infected erythrocytes [49]. These defects reduced actin polymerization and as a result reduced protein trafficking from the Maurer's clefts (a parasite-derived organelle). The defects are less in infected red cells containing Hb AS, Hb AC, and Hb F compared with infected red cells containing Hb AA [50]. Interestingly, similar defects in actin polymerization can be shown when normal Hb AA erythrocytes are exposed to a transient moderate oxidant stress before invasion of red blood cells, suggesting that the molecular species that interfere with actin reorganization and cytoadhesion are irreversibly oxidized products, such as ferryl hemoglobin, hemichrome radicals, or ferryl heme [50].

●Reduced cytoadherence – Infected red blood cells from sickle trait individuals (Hb AS) demonstrate reduced in vitro adhesion to microvascular endothelial cells and blood monocytes relative to adhesion of infected Hb AA red blood cells. Hb AS red cells also show reduced expression on their surface of the variant antigen Pf-EMP-1 (P. falciparum erythrocyte membrane protein-1), the parasite's major cytoadherence ligand and virulence factor, and reduced adhesion to CD36 and endothelial protein C receptor (EPCR) [51,59]. Sickle red blood cells display altered endothelial cell adhesion and decreased adhesion resulted in less endothelial cell activation [60].

Furthermore, reduced cytoadherence in malaria-infected red blood cells compared to that seen in infected cells from patients with sickle cell trait and alpha⁺ thalassemia negativity is entirely consistent with epistasis between these two traits [52].

The relative importance of these phenomena in patients with malaria is unclear. There is conflicting experimental evidence for enhanced phagocytosis or retention of ring-stage-infected Hb AS red blood cells compared with ring-stage-infected Hb AA red blood cells. There are consistent experimental data showing reduced expression of the variant antigen, Pf-EMP-1, the parasite's major cytoadherence ligand of infected red blood cells. It is possible that enhanced clearance of ring-stage-infected red cells and reduced adhesion of mature infected red cells both contribute to the remarkable protection due the Hb AS phenotype.

●Immune mechanisms – A series of intriguing observations suggests that the protection of people with sickle cell trait may be mediated, at least in part, by enhancing the acquired immune response to malaria [53,61,62]. Such an explicit immune-mediated mechanism(s) would explain observations that the effect of Hb AS on parasite density was strongly age-dependent, only beginning after two years [53].

Two studies have found significantly raised gamma globulin levels in young Hb AS children [63,64]. More specifically, children with the Hb AS phenotype had higher levels of anti-sporozoite antibodies and agglutinating antibodies to variant surface antigens [26] and significantly increased lymphoproliferative responses to malaria antigens [65]. However, the antibody response against a microarray of P. falciparum proteins showed no significant difference in antibody responses in children with Hb AS and Hb AA controls [66]. It remains possible that Hb AS and Hb AC may modulate other protective immune responses.

There are some direct epidemiological data suggesting that protection mediated by sickle hemoglobin may involve the accelerated acquisition of malaria-specific immunity. The age-specific protection afforded by Hb AS against clinical malaria in children living on the coast of Kenya increased from 20 percent in the first two years of life to a maximum of 56 percent by the age of 10 years, returning to 30 percent in children more than 10 years old [67].

There are no equivalent data for the other hemoglobinopathies; a small study in Gambian children with thalassemia trait showed no differences in their immune responses to malaria compared with controls [68].

The mechanism of an enhanced immunologic response has not been established either in vitro or by ex vivo clinical studies. While infected variant erythrocytes may be able to stimulate immune responses, early clearance of infected erythrocytes at the ring stage of development or reduced adhesion of mature infected red blood cells may prevent immune responses against malaria parasites by several mechanisms:

●Removal of ring-stage parasites would reduce production of malaria pigment formed by the digestion of hemoglobin in the host erythrocytes and subsequent dysregulation of monocyte function [69].

●Removal of ring-stage parasites or reduced expression of the variant antigen PfEMP-1 on mature infected red blood cells would reduce adhesion of infected erythrocytes to myeloid dendritic cells and macrophages, decreasing effects on innate and acquired immune responses [70,71].

Mouse models — Mice are susceptible to several species of Plasmodia, and a transgenic mouse model expressing sickle cell disease has allowed new approaches to dissecting the protective mechanisms of malaria [7,72-74]. In studies of sickle transgenic mice, P. chabaudi adami, P. berghei, and the P. yoelii strains were used in nonlethal and lethal infections. In these studies, a transgenic mouse line expressing both the sickle gene and human alpha globin genes was used. To increase the relative expression of sickle globin, the transgenic mice were bred to homozygosity for an alpha thalassemia mutation, resulting in an animal model intermediate in severity between sickle cell trait and sickle cell anemia.

When these mice were infected with two species of rodent malaria, they showed a diminished and delayed increase in parasitemia compared with controls [7]. This benefit in P. chabaudi adami infection was completely prevented by splenectomy, suggesting that enhanced removal of malaria-infected Hb AS cells by the spleen, at least in this model, is required to mediate protection from malaria.

As in humans, the protection afforded by Hb S is dose-dependent in transgenic mice. This was illustrated in a study that evaluated the response of three transgenic models, expressing 39, 57, and 75 percent Hb S, to challenge with a virulent strain of P. yoelii that is lethal and appears to cause cerebral malaria [75]. Protection was in direct proportion to the level of expression of the sickle gene:

●Low expressors (39 percent Hb S) and controls (no Hb S) rapidly succumbed to the infection.

●Seven of nine intermediate expressors (57 percent Hb S) initially survived, but all died at day 6.

●The majority of high expressors (75 percent Hb S) survived; those who ultimately died survived up to 16 days.

Among mice in the last two groups, malarial parasites were found almost exclusively in reticulocytes, suggesting that there is greater resistance to malarial infection in mature erythrocytes expressing Hb S.

There is also evidence for modulation of the innate immune response to malaria. One murine model of sickle cell anemia with significant steady state hemolysis has shown that these mice are resistant to experimental cerebral malaria (ECM). This protective effect was attributed to induction of heme oxygenase-1 (HO-1) in hematopoietic cells, via a mechanism involving the transcription factor NF-E2-related factor 2 (Nrf2) and generation of carbon monoxide (CO) as a byproduct of heme catabolism by HO-1. This prevented further accumulation of circulating free heme, activation of the immune response, and activation and/or expansion of pathogenic CD8⁺ T cells. It is unclear if these mechanisms are present in human infections [76].

Sickle cell disease — It is widely believed that malaria is a major risk factor for death among children with sickle cell disease (SCD), although there is no clear evidence that children with SCD are at greater risk from malaria than children without SCD [77,78]. Children with Hb SS are no more likely to have uncomplicated malaria or malaria complicated by a range of well-described clinical features of severity than controls. Nevertheless, mortality was considerably higher among children with Hb SS than controls with Hb AA or Hb AS children hospitalized with malaria [79]. Children with Hb SS are at higher risk of readmission to the hospital after severe malaria than children with Hb AA [80].

Similar results were reported from Tanzania, where parasitemia during the hospitalization of children with SCD was associated with both severe anemia and death [81]. Indeed, intermittent preventive treatment with sulfadoxine-pyrimethamine in patients with Hb SS may significantly reduce patients' complaints and blood requirements, but not vaso-occlusive crises and hospitalization [82,83].

Conclusions — People with sickle cell trait are partially protected from death caused by P. falciparum malaria through several possible mechanisms. The first line of defense is a dramatic acceleration of sickling rates in parasitized sickle cell trait cells that facilitates their removal from the circulation. Parasites that escape this process show impaired growth when the host cells become hypoxic and adhere to the endothelium of venules. Second, ring-stage-infected red blood cells from people with Hb AS undergo increased phagocytosis compared with infected red blood cells from people with Hb AA. These effects may lead to an accelerated acquisition of immunity to malaria. It is unclear if any of these mechanism(s) is significant in vivo.

Intriguingly, some parasite genotypes are found more frequently in people with Hb AS than people with Hb AA, suggesting some parasite traits confer a selective advantage [84]. The Hb S-associated alleles include nonsynonymous variants in the acyl-CoA synthetase family member PfACS8 on chromosome 2 and two other regions of the parasite genome [84]. Understanding the biological mechanisms that confer advantages for some parasite genotypes may help clarify the mechanisms by which Hb AS trait is protective against malaria.

It has been generally thought that people with sickle cell anemia (Hb SS) remain susceptible to malaria, although the relative risk of malaria in Africans with sickle cell anemia has not been established. While patients with sickle cell anemia may be less likely to die from cerebral or hepatic malaria, they may succumb to other effects of infection (eg, volume depletion, acidosis, cytokine release).

HEMOGLOBIN C — The hemoglobin C (Hb C) gene has its maximum frequency in northern Burkina Faso, and the prevalence decreases concentrically in surrounding areas. A case-control study involving 4348 Mossi individuals from Burkina Faso suggested a 29 and 93 percent reduction in risk of clinical malaria in Hb C heterozygotes (Hb AC) and homozygotes (Hb CC), respectively [85]. These data were supported by a case-control study in Ghana showing that children with Hb AC and Hb CC genotypes were protected from severe malaria [86]. However, the data for protection against mild malaria for Hb AC heterozygotes are less clear, as two prospective cohort studies from Mali have shown inconsistent results. A study from Bamako in Mali showed that mild malaria incidence was higher in Hb AC children than in Hb AA children (adjusted incidence rate ratio 1.15; 95% CI 1.01-1.32) [87], while a similar study in the town of Bandiagara in a younger-aged cohort showed fewer episodes of clinical malaria, and a lower cumulative parasite burden in children with Hb AC compared to controls with Hb AA [88]. Moreover, Hb AC was associated with higher newborn birthweight among women with pregnancy-associated malaria, conferring a survival advantage for higher-birthweight newborns from Hb AC mothers [89].

Mechanisms of protection — In vitro studies in oxygenated Hb CC cells have shown the following results [42]:

●No abnormality in parasite invasion.

●Normal growth during the first growth cycle but a markedly reduced number of ring forms following the schizont stage, with degenerated schizonts observed on day 4. This defect was not affected by deoxygenation or an increase in the extracellular potassium concentration, excluding a potassium leak-dependent mechanism.

●Marked resistance to osmotic lysis compared with parasitized normal cells. Thus, these cells cannot burst and release merozoites in a normal fashion.

An important observation that remains unexplained is that Hb C trait red cells sustain parasite growth as well as do normal red cells. However, there is good epidemiological evidence that there is a protective effect of the heterozygous (Hb AC) state, especially in regard to cerebral malaria [88,90].

Hb C modulates the cell surface properties of P. falciparum-infected erythrocytes by reducing expression of the variant antigen (Pf-EMP-1) [91]. As a result, infected red blood cells with Hb AC or Hb CC show reduced adhesion to endothelial cells expressing CD36 and intercellular adhesion molecule-1 (ICAM-1), reduced rosetting with uninfected red blood cells, and reduced agglutination in the presence of pooled sera from malaria-immune adults. In malaria-infected red blood cells, the presence of Hb C may inhibit actin polymerization and the production of Maurer's clefts, both of which are necessary for directing parasite-encoded proteins to the surface of infected red blood cells [48]. Further studies have extended these findings and shown that the kinetics of protein trafficking from the parasite to different host cell compartments, including the cytoplasm, the Maurer's clefts, and the plasma membrane is delayed, slower, and with reduced amounts of exported protein in parasitized Hb AS and Hb AC erythrocytes [92].

These data strongly suggest that sequestration of Hb C-containing erythrocytes would be diminished compared with Hb AA-infected red blood cells and thus represent a potent mechanism of protection.

HEMOGLOBIN SC DISEASE — In culture, oxygenated hemoglobin (Hb) SC cells (ie, red cells from patients who are doubly heterozygous for the presence of hemoglobins S and C) are indistinguishable from normal cells as a host for Plasmodia. However, the parasite rapidly dies when Hb SC cells are partially deoxygenated [93]. Why this occurs is unclear.

Mechanisms of protection — Possible mechanisms whereby patients with Hb SC disease might be protected against malaria include [94]:

●Lowering of the intra-erythrocyte pH by parasite metabolism, inducing more deoxy Hb S polymerization in a red cell with high mean corpuscular hemoglobin concentration (MCHC) due to the presence of Hb C.

●Increased crystallization of Hb C induced by the presence of Hb S, creating an inadequate substrate for the parasite's proteases.

There is, at least in the United States, little mortality from Hb SC disease before reproductive age [95]. Even in the extreme conditions of some parts of sub-Saharan West Africa, it is likely that individuals with Hb SC disease have a significantly higher fitness than individuals with Hb AA because of their resistance to malaria. They may also have a higher fitness than individuals with Hb C trait, as demonstrated by the intensity of parasite growth inhibition in vitro.

A model of this type would explain the introduction of an additional advantageous hemoglobin gene into a population that already has one or more genes that provide malaria resistance. Combined heterozygotes might compound the mechanisms of resistance, producing a more fit individual in a malarious region.

HEMOGLOBIN E — Hemoglobin (Hb) E, a mutation of the beta globin chain, which is also associated with reduced expression, is the most frequent hemoglobin structural mutation in Southeast Asia [96]. The highest frequency of this gene is observed in the "Hb E triangle" where the frontiers of Cambodia, Laos, and Thailand converge. In the central region of Indochina, red cell genetic defects (eg, Hb E, thalassemia, and glucose-6-phosphate dehydrogenase [G6PD] deficiency) are so common that only about 15 percent of the population has "normal" red cells [97]. (See "Hemoglobin variants including Hb C, Hb D, and Hb E", section on 'Hb E'.)

Early epidemiological studies suggested a connection between this abnormal hemoglobin and malaria [96]. In addition, carriers of Hb E (both disease and trait) had significantly higher levels of antimalarial antibodies than individuals with Hb AA from the same geographic areas [98].

While there is no direct evidence for the protective effect of Hb E in malaria, the pattern of linkage disequilibrium surrounding the beta globin locus in Thailand suggests a single origin of Hb E arising between 1200 to 4400 years ago and a rapid increase in allelic frequency [99].

Mechanisms of protection — Initial in vitro studies of the relationship between P. falciparum and Hb E-containing cells demonstrated a moderate decrease in growth of P. falciparum in Hb E disease cells but not Hb E trait cells [100]. A subsequent report showed diminished growth of parasites in both Hb E trait and Hb E disease cells that was most pronounced as the concentration of Hb E increased; this effect was maximal at 20 percent oxygen and present but decreased at 5 percent oxygen [98].

Additional studies suggest that red cells from individuals with Hb E trait have a membrane "difference" that renders the majority of the red cell population relatively resistant to invasion by P. falciparum, preventing development of heavy parasite burdens [101]. In addition, Hb E is somewhat unstable and is capable of generating free radicals and inducing oxidative damage to the red cell membrane [102]. The oxidant activity of Hb E may explain the following observations:

●Vitamin C, an antioxidant, partially inhibits the Hb E-associated reduction in parasite growth in 20 percent oxygen cultures [98].

●Parasitized Hb E disease and Hb E trait erythrocytes are phagocytized to a greater extent by normal human monocytes than infected erythrocytes from individuals with Hb AA [4]. This phenomenon is particularly prominent in late trophozoites and schizonts, suggesting that the surface of parasitized Hb E-containing red cells is modified compared with normal red cells.

NEONATAL RED CELLS AND HEMOGLOBIN F — The growth of P. falciparum is diminished in red cells containing fetal hemoglobin (Hb F), even though parasite invasion may be increased in umbilical cord red cells, which have a high concentration of Hb F [103,104]. Reduced parasite growth appears to be due directly to Hb F, since it can be demonstrated in all red cells containing Hb F. These include cord blood cells containing a majority of Hb F, red cells from infants' F cells (which contain about 20 percent Hb F), and red cells from adults homozygous for hereditary persistence of fetal hemoglobin (HPFH), whose red cells contain almost 100 percent Hb F [104]. However, other groups have not found such growth defects [105].

Mechanisms of protection — Initial studies suggest that the decrease in parasite growth associated with the presence of Hb F may be mediated by an increase in intraerythrocytic oxidative stress [106]. This mechanism was studied directly in transgenic mice with 40 to 60 percent Hb F [107]. The mice were infected with three types of rodent malaria and the following results were noted:

●In mice infected with P. chabaudi adami, which causes a nonlethal infection mainly in mature red cells, there was a more rapid rise in parasitemia than in control mice, consistent with the findings noted above [103,104], but the parasitemia was cleared more rapidly.

●In mice infected with P. yoelii 17XNL, which induces a nonlethal infection that primarily consists of invasion of reticulocytes, there was a reduction in parasitemia that was not reversed by prior splenectomy. This is different from the importance of the spleen in the protective effect of Hb S, as described above [7].

●The benefit was greatest in mice infected with P. yoelii 17XL, a lethal variant that produces a syndrome resembling cerebral malaria caused by P. falciparum in humans [108] and almost invariably causes death in one to two weeks. While control mice died between 11 to 23 days, transgenic mice cleared the infection by day 22 and survived. This effect was not mediated by the spleen, since similar results were seen in splenectomized animals.

●Light microscopy revealed that intraerythrocytic parasites developed more slowly in Hb F-containing erythrocytes. Hb F was digested only half as well as Hb A by malarial hemoglobinases (recombinant plasmepsin II), which could explain the slowing of parasite growth.

Thus, resistance to malaria of normal neonates and infants in the first six months of life can be explained by a double mechanism: increased malaria invasion rates in Hb F-containing cells, resulting in fewer parasites available for invasion of the Hb A-containing cells; and slowed growth within the Hb F cells [104]. These factors may also contribute to the advantages of being a carrier of thalassemia, since this disorder is associated with a significant slowing of the postnatal switch from Hb F to Hb A. Protection during the first six months of life is critical, since humoral and cellular defenses are not yet effective during this period.

THALASSEMIA — Both alpha thalassemia and, to a lesser degree, beta thalassemia are protective against malarial infection, although fewer studies support the selective advantage of thalassemia compared with other red cell polymorphisms.

The initial evidence supporting malaria protection by both forms of thalassemia was derived from population genetic studies. Globally, the thalassemias are the most common single gene disorders thus far described [109]. Overall, gene frequencies >0.10 are the norm in tropical populations, whereas frequencies are somewhat lower in the subtropics and rare in the temperate zones. Moreover, isolated examples of extreme frequencies have been described in particular ethnic groups, particularly in certain tribes in India and Nepal [110-113]. In one of these tribal groups, the Tharu people of Nepal, the alpha thalassemia gene frequency reaches 0.78 [114].

While the global distribution of the thalassemias provides reasonable evidence for malaria protection, the probability that malaria was responsible for this selection was further supported by micro-epidemiological data from Italy and the Pacific. In a series of classic studies conducted in the 1960s, a cline (or gradient) in the population frequencies of beta thalassemia in Sardinia correlated with altitude [115]. Although malaria was no longer endemic in Sardinia when these studies were conducted, historically, the incidence of malaria was known to have correlated closely with altitude. The favored hypothesis was that the cline represented selection for the thalassemic beta globin gene under pressure from malaria.

Support for this hypothesis has since come from the Pacific. While the gene frequency for beta thalassemia followed a similar correlation with altitude in Papua New Guinea [116], it was the data for alpha thalassemia collected from communities throughout Melanesia that was perhaps the more dramatic. These data showed that alpha thalassemia was found in all malaria-exposed populations and at gene frequencies that were proportional to the incidence of malaria based on historical reports.

A cline in the alpha thalassemia haplotype frequency was seen both from north to south and with increasing altitude, each of which are paralleled by a cline in the historical incidence of malaria. In addition, the genetic deletions responsible were numerous and regionally specific. Further, population frequencies of other "neutral" genetic markers, including the gamma globin haplotypes, showed no such correlation [117,118]. A similar conclusion was reached in a study of children resident in 13 villages in the Eastern Arc Mountains in Tanzania, where P. falciparum transmission intensity is closely correlated with altitude [119].

Alpha thalassemia protects children from severe malaria [120].

Additional evidence for a protective effect of the thalassemia traits is discussed below.

Oxidant injury — It has been proposed that, in the thalassemias (ie, alpha and beta thalassemia, Hb E and Hb Constant Spring), oxidant injury is a prominent factor in parasitized thalassemic red cells and contributes to the protection against severe malarial infection. Parasitized red cells are under substantial oxidant challenge [121], an effect that may be enhanced by the release of superoxide anions by effector T cells upon binding to infected red cells [122,123]. In addition, the red cell membrane in thalassemic cells undergoes oxidative damage initiated by the binding of excess globin chains, mostly in the form of hemichromes. (See "Pathophysiology of thalassemia".)

The combination of intrinsic thalassemic injury and the effect of malarial infection produces a red cell with less viability. These changes permit the preferential destruction and removal of thalassemic parasitized red cells.

Intraerythrocytic multiplication — It is possible that hemoglobinopathic erythrocytes reduce the intraerythrocytic multiplication of P. falciparum, potentially delaying the development of life-threatening parasite densities until parasite clearing immunity is achieved [124].

Beta thalassemia — The potential protective effect of beta thalassemia trait was illustrated in a population survey in northern Liberia [24]. There was an increasing frequency of thalassemia trait with increasing age, suggesting that carriers had increased survival. Although P. falciparum prevalence rates were similar in normal and beta thalassemia trait children, the latter group had lower parasite densities. Using a parasite density of ≥1 x 10⁹/L to indicate a potentially lethal infection, the relative risk was 0.45 in children between the ages of one and four with beta thalassemia trait when compared with those without thalassemia.

Mechanisms of protection — Both diminished parasite growth and altered function of infected red blood cells may contribute to the protection against malaria in beta thalassemia. In initially-performed culture studies, parasites grew normally in thalassemia trait red cells, but parasites were more susceptible to oxidants than were parasites growing in normal cells [106]. However, the inability to demonstrate impaired parasite growth in this and other studies [125] may have been due to the use of culture media with an abundance of amino acids essential for parasite growth that are usually obtained by the digestion of hemoglobin by malarial hemoglobinases [126]. Such an abundance of amino acids in the culture media would mask the limitations encountered by the parasite in the hypochromic (ie, hemoglobin deficient) red cells of individuals with thalassemia.

The impairment in parasite growth that has been demonstrated in other studies varies with the type of thalassemic erythrocyte. This impairment is less prominent in heterozygotes with beta thalassemia than in alpha thalassemia, the alpha thalassemia variant Hb H/Constant Spring, or combined beta thalassemia/Hb E [126,127].

The mechanisms involved in diminished parasite growth in beta thalassemic cells are not well understood, but persistence of Hb F plays a contributory role in some patients [103,104] In addition, oxidant injury may promote the preferential removal of parasitized thalassemic red cells (see 'Oxidant injury' above).

Transgenic mice have contributed to our understanding of the relationship between beta thalassemia and malaria. Beta thalassemic mice have lower and delayed peak parasitemia after infection with P. chabaudi adami, which has no preference for immature red cells, but not with P. berghei, which preferentially invades reticulocytes [128]. The protective effect against P. chabaudi adami was reversed by transgenic correction with the normal human beta globin gene.

In addition to reduced parasite growth, beta thalassemic erythrocytes infected with P. falciparum show marked reductions in the tendency to cytoadhere and to form rosettes [129]. These abnormalities are associated with diminished expression of parasite antigens on the surface of infected thalassemic red cells in the trophozoite or schizont stage, but not in the ring form stage.

Malaria-infected erythrocytes from people with beta thalassemia trait are susceptible to enhanced phagocytosis of ring forms of the parasite. Membrane-bound hemichromes, autologous immunoglobulin G (IgG) and complement C3c fragments, aggregated band 3, and phagocytosis by human monocytes were increased in ring forms of the parasite developing in beta thalassemia trait erythrocytes compared with control infected erythrocytes [6]. Enhanced phagocytosis of ring-parasitized mutant red blood cells may represent the common mechanism for malaria protection in nonimmune individuals affected by beta globin variants, while individuals with alpha thalassemia trait are likely protected by a different mechanism.

Alpha thalassemia — The overall distribution of alpha thalassemia in malaria endemic areas may be explained by negative epistasis with sickle cell trait. Thus, while both sickle cell trait and alpha thalassemia offer protection against malaria, this protective effect is lost when alpha thalassemia and sickle cell anemia are co-inherited. This phenomenon could explain the failure of alpha thalassemia to reach fixation in any population in sub-Saharan Africa [31], as well as why the sickle cell gene is uncommon in the Mediterranean, where the genes for thalassemia are common [130].

In three studies, one from the South Pacific [131], and the other two from Africa [31,120], the relative risk of severe malaria was significantly reduced in carriers of alpha thalassemia trait. In PNG, compared with controls, the risk of severe malaria was 0.40 (95% CI 0.22-0.74) in alpha thalassemia homozygotes and 0.66 (95% CI 0.37-1.20) in heterozygotes. Moreover, the risk of hospital admission with infections other than malaria was reduced to a similar degree in homozygous (0.36; 95% CI 0.22-0.60) and heterozygous (0.63; 95% CI 0.38-1.07) children [131].

Ghanaian children with alpha thalassemia were protected from severe malaria; heterozygous alpha thalassemia was observed in 32.6 percent of controls but in only 26.2 percent of cases (odds ratio [OR] 0.74, 95% CI 0.56-0.98) [120]. In Kenya, the prevalence of both heterozygous and homozygous alpha thalassemia was reduced in patients with severe malaria compared with controls (adjusted ORs 0.73 and 0.57, 95% CIs 0.57-0.94 and 0.40-0.81) respectively [132].

A small study focusing on mild malaria has shown that alpha thalassemia is associated with a reduced risk of uncomplicated malaria episodes in children in northeastern Tanzania [133]. This advantageous effect seemed to be more predominant in children >5 years of age, although large longitudinal studies are required to derive reliable data on the protection for any genetic trait with age.

Large scale detailed studies of the effect of alpha thalassemia on specific syndromes of severe malaria have suggested a potential mechanism of protection. However, the presence of alpha thalassemia showed protection from severe malarial anemia and severe nonmalarial anemia. The incidence rate ratios for alpha thalassemia heterozygotes and homozygotes combined compared with Hb AA children were 0.33 (95% CI 0.15-0.73) for severe malaria anemia and 0.26 (95% CI 0.09-0.77) for severe nonmalarial anemia [134]; these findings were confirmed in a larger study [10]. Heterozygous alpha thalassemia trait has also been associated with reduced risk of asexual parasitemia (primarily submicroscopic) in children but not in adults or pregnant women in Ghana (OR: 0.52; 95% CI 0.28-0.97) [135].

Mechanisms of protection — The protection against severe malaria associated with alpha thalassemia may be mediated in part by increased susceptibility to infection with the nonlethal P. vivax, particularly in young children, thereby inducing limited cross-species protection against subsequent severe P. falciparum infection [127,136,137]. This possibility was illustrated in a study of the epidemiology of malaria in Vanuatu in the northeast Pacific [137]. The prevalence of uncomplicated malaria (P. falciparum and P. vivax) and of splenomegaly (commonly associated with malaria infection) was increased among children under the age of five years with alpha thalassemia; this was not seen in older children.

Culture studies suggest impaired P. falciparum growth within infected cells from patients with alpha thalassemia or the alpha thalassemia variants Hb H and Hb Constant Spring [5,126,127,138]. This inhibitory effect was seen only in severe alpha thalassemia (ie, those missing three of the four normal alpha chain genes), and not in those missing only one or two alpha globin genes, and was independent of the presence of Hb F [138]. Another contributing factor in Hb H and Hb Constant Spring may be increased phagocytosis of infected red cells [5]. Such an extrinsic mechanism cannot be detected in culture.

Reduced expression of red cell complement receptor 1 (CR1), responsible for red cell rosetting of infected and non-infected red cells, has been associated with alpha thalassemia and may confer protection against severe malaria via this mechanism. Low CR1 expression on reticulocytes reduces the ability of P. vivax to invade, and the selection of CR1 alleles associated with low receptor expression suggests that CR1 levels are important host factors mediating P. vivax invasion capacity [139]. (See "Pathogenesis of malaria".)

Infected red cells from people with alpha thalassemia appear to have reduced expression of the variant antigen Pf-EMP-1 and subsequently reduced adherence of infected red blood cells to microvascular endothelial cells (MVECs) and monocytes in vitro [140]. Such a mechanism may reduce sequestration and the progression to severe disease.

Some of the protective effect of homozygous alpha thalassemia on severe anemia may be explained by the increased erythrocyte count and microcytosis, allowing a greater reduction in erythrocyte count than children of normal genotype during malaria infection before the hemoglobin level falls to cause severe anemia (eg, total hemoglobin concentration <5 g/dL) [141,142].

Interaction with haptoglobin genotypes — Whether the risk of severe malaria is altered by haptoglobin (Hp) genotype is unclear, with four studies of this question producing conflicting results. However, one study uncovered a significant epistatic interaction between alpha thalassemia and Hp genotype that may explain why studies that do not examine both genotypes could give conflicting results. Whereas Hp2-1 inherited in combination with alpha⁺ thalassemia offered significant (37 percent) protection against severe malaria, such protection was considerably reduced (13 percent protection) when inherited in combination with a normal alpha globin genotype. Moreover, in those with alpha thalassemia, protection was lost altogether when inherited in combination with Hp2-2 [143]. Indeed, there was a non-significant trend towards increased susceptibility in those who inherited Hp2-2 and alpha thalassemia. The available data suggest that complex epistatic interactions arise from the fact that the Hp2-2 molecule is significantly less able to quench hemoglobin-iron mediated oxidative stress, and is further associated with a shift towards a pro-inflammatory Th1 cytokine response. The available data suggest that Hp2-1, but not Hp1-1 or Hp2-2, confers a selective advantage through protection from malaria but only in the presence of alpha thalassemia. The data require confirmation in other groups and/or further mechanistic studies before firm conclusions can be drawn.

SUMMARY — A number of human genes share a distribution that is strikingly similar to that of malaria. It has now been confirmed that many of these traits provide substantial protection against the disease. (See 'Malaria protection and inherited factors' above.)

The following hemoglobin (Hb) disorders have been shown to be protective of malarial infection and/or to reduce disease severity:

●Sickle cell trait (see 'Falciparum malaria and hemoglobin S' above)

●Hb C trait and Hb C disease (see 'Hemoglobin C' above)

●Hb SC disease (see 'Hemoglobin SC disease' above)

●Hb E trait and Hb E disease (see 'Hemoglobin E' above)

●Alpha and beta thalassemias (see 'Thalassemia' above)

Fetal Hb (Hb F) is also protective. (See 'Neonatal red cells and hemoglobin F' above.)

A wide variety of potential mechanisms of protection against malaria continue to be described, but it is not clear which of these are important in vivo.

Inherited abnormalities of red blood cell surface antigens and cytoskeletal proteins, which may also protect against malarial infection, are discussed separately. (See "Protection against malaria by abnormalities in red cell surface antigens and cytoskeletal proteins".)

ACKNOWLEDGMENT — We are saddened by the death of Stanley L Schrier, MD, who passed away in August 2019. The editors at UpToDate gratefully acknowledge Dr. Schrier's role as Section Editor on this topic, his tenure as the founding Editor-in-Chief for UpToDate in Hematology, and his dedicated and longstanding involvement with the UpToDate program.