Pathogenesis of anemia in malaria: a concise review
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- Ghosh, K. & Ghosh, K. Parasitol Res (2007) 101: 1463. doi:10.1007/s00436-007-0742-1
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Anemia is a common complication in malarial infection, although the consequences are more pronounced with Plasmodium falciparum malaria (Ghosh, Indian J Hematol Blood Tranfus 21(53):128–130, 2003). Anemia in this infection is caused by a variety of pathophysiologic mechanisms, and in areas where malaria infection is endemic, co-morbidities like other parasitic infestations, iron, folate and Vitamin B12 deficiency, deficiency of other nutrients, and anemia, which is aggravated by anti-malarial drugs both through immune and non-immune mechanisms, are important considerations. In different endemic areas, β-thalassemia, α-thalassemia, Hb S, Hb E, G6PD deficiency, or ovalocytosis in different proportions interact with this infection. Finally, aberrant immune response to repeated or chronic falciparum malarial infection may produce tropical splenomegaly syndrome, a proportion of which show clonal proliferation of B lymphocytes. Cooperation between chronic malarial infection and infection with E-B virus infection in producing Burkitt’s lymphoma is well known. In this review, the fascinating and multifaceted pathophysiolgoy of malarial anemia has been discussed.
Approximately 300–500 million people are infected with malaria all over the world every year (Snow et al. 1999), and a substantial proportion of these patients live in parts of the world where malaria is endemic, leading to chronic infection or repeated infection. In some parts of Africa at any point of time, almost 100% children show asexual stage of malarial parasite in their blood (Githeko et al. 1993).
Anemia is a common complication in both acute and chronic malaria. In one extreme acute malarial infection in a severely G6PD-deficient subject, when exhibited drugs like Quinine can produce life-threatening acute intravascular hemolysis with severe anemia and acute renal failure (Blackwater fever), and on the other extreme, a chronically infected patient may have mild chills and splenomegaly and mild to moderate anemia. In between lies all shades of anemia with malarial infection caused by diverse pathophysiologic mechanisms. In each case, multiple mechanisms are at work, but anemia in any individual case may mainly be cased through one or two predominant pathophysiological mechanisms (Wickramasinghe and Abdalla 2000).
Anemia of chronic disorder
Anemia of chronic disorder is a very well-known entity. Chronic infection and other chronic disorders cause (Means 2003) this type of anemia. Classically, anemia of chronic disorder is characterized by moderate to mild normochromic normocytic anemia or hypochronic microcytic anemia associated with hypoferrimia and raised levels of inflammatory protein like fibrinogen, orosomucoid, coupled with low total iron-biding capacity, low tranferrin levels and low reticulocyte count. Broadly, this kind of anemia is hypogenerative along with mild to moderate shortening of red-cell life span. So long as the chronic disorder persists, giving iron therapy does not correct the anemia. Interleukin (IL)-1, tumor necrosis factor alpha (TNF-α) and IL-6 cytokines, which are also raised in malarial infection, are believed to cause this kind of anemia (Abdalla 1990; Woodruff et al. 1979).
Direct invasion of red cells
As a part of the life cycle, intraerythrocytic schizogony of malaria parasites takes place inside the red cells. Plasmodium falciparum has a schizogony cycle of 24–36 hours, and at the end of this cycle, the merozoites come out by disrupting the red cells. P. falciparum normally can invade red cells of any age, hence the fraction of RBC infected with the parasite could be very high in contrast to Plasmodium vivax infection, which tends to infect smaller proportion of young and larger red cells including reticulocytes. Among the important causes of reticulocytopenia in malarial infection is direct invasion by the parasite in addition to the aregenerative anemia of chronic infection and dyserythropoiesis.
Over millions of years in its quest for perfect receptor protein for attachment, various species of malaria parasites developed one or several ligands in its body to attach to the red cell membrane and enter inside the cell. Duffy binding proteins, Duffy-like antigen-binding proteins, reticulocyte binding ligands, ligands to bind glycophorin C and D are few such examples (Mayer et al. 2001; Chitnis 2001). Merozoitis destroy the red cell membranes causing its rupture by its own secreted protease and is an important cause for direct hemolysis.
Iron shunting for utilization by the parasites
In the pathogenesis of anemia of chronic disorder, hypoferrimia was found to be an extremely important finding. This hypoferrimia is brought about (1) by locking the iron in the macrophage stores, (2) by synthesizing iron-binding proteins of higher affinity by inflammatory cells compared to transferrin and (3) by reducing the amount of transferrins synthesized in the liver. The evolutionary idea of this process was to deny the growth-promoting iron to siderophilic organizers of which malaria parasite is undoubtedly one of them.
Malaria parasite needs lots of iron for its own life cycle, and it manages to extract the iron from the host by inserting parasite-specific transferrin-like receptors on the host red cell membrane. Malaria parasites can therefore cause additional iron deficiency in the host (Oppenheimer 1989). This pathophysiology as the causation of anemia may be more pronounced where nutritional iron deficiency is extremely common, as in some parts of India where >60% of the women and >30% of the men are iron deficient.
Glucose 6 phosphate dehydrogenase deficiency hemoglobinopathies and South Asian ovalocylosis
Several lines of evidence have shown that G6PD-deficient red cells, inheritance of hemoglobinopathies like α-thalassemia, β-thalassemia, Hb S and Hemoglobin E tends to protect an individual from life-threatening malarial infection or death by various mechanisms (Luzzatto et al. 1969; Ayi et al. 2004; O’Donnell et al. 1998). This protection is available at some cost, i.e. increase in severity of anemia when malarial infection takes places in carriers of these genetic traits. These traits are also present at higher and at a mix of varying frequencies where malarial infections are known to be endemic for centuries. G6PD deficiency deserves special mention here because inheritance of this deficiency may cause life-threatening acute intravascular hemolysis along with hemoglobinemia and acute renal failure either with severe malarial infection and/or when an oxidant antimalarial drug like Quinine or Primaquin is given. This condition known as Blackwater fever happens in persons who inherit Mediterranean type of G6PD variants where the enzyme activity is near zero and reticulocytes are also devoid of this enzyme, as a result of which hemolysis continues in spite of brisk reticulocyte response so long as the oxidant drug effect continues. In African population where G6PD deficiency is of A type, hemolysis due to infection or due to oxidant drugs is episodic and affects only older G6PD-deficient cells. The hemolysis stops as soon as young red cells and reticulocytes which contain adequate amount of this enzyme come out in peripheral blood even though the offending drug is continued.
Hypersplenism and dynamic changes in splenic function
Malarial infection produces intense stimulation of monocyte macrophage system leading to hypersplenic state even when the malaria is acute. Hypersplenism continues to be operative 4–6 weeks after malaria parasites are cleared from blood (Looareesuwan et al. 1987). Even non-parasitised RBCs have changes in the membrane which predispose them to removal by the spleen. The osmotic fragility of non-parasitised RBCs in malaria infection is increased resulting in significant hemolysis of non-parasitised RBC. In chronic malaria, hypersplenism could be significant mechanism in causation of anemia (Woodruff et al. 1979; Roberts et al. 2005).
Haemophagocytosis in occasional patients may cause significant anemia and cytopenia in a small subset of patients with malarial infection (Ohno et al. 1996; Clark et al. 2006). It is not clearly known whether in these patients there is underlying immunodeficiency or EB virus infection to drive the haemophagocytosis or if it is because of inappropriate secretion of cytokines like TNF-α in the background of proper genetic background of the host.
Dyserythropoiesis and impaired erythropoietesis response
In malarial anemia, reticulocytopenia is associated with high levels of erythropoietin secretion. High level of erythropoietin in malaria is caused by high levels of hypoxia inducing factor 1 (HIF-1) induced by a combination of high levels of TNF-α (Sandau et al. 2001). Significant dyserythropoiesis as a cause of malaria has been shown to occur by several workers in both P. falciparum and P. vivax infection (Srichaikul et al. 1967; Abdalla 1990; Wickramasinghe et al. 1989).
Dyserythropoiesis in malarial infection was found to correlate with deficient interleukin-12 production (Mohan et al. 1998). Erythroid colony like BFU-E and CFU-E production is also significantly inhibited from the bone marrow of malaria-infected patients or in experimental animals (Abdalla et al. 1988; Martiney et al. 2000).
Cytokine dysregulation in malarial infection
Acute malarial infection as well as chronic malaria produces intense cytokine dysregulation with tremendous increase in IFNγ, IL-6, TNF-α IL-1, MIF and HIF-1. The changes in P. falciparum infection is common to systemic inflammatory state. However, there is also significant underproduction of IL-10 and IL-12. Severe anemia in malaria is usually associated with low levels of IL-10 and IL-12 (Weatherall et al. 2002; Harpaz et al. 1992), Othoro et al. 1999; Perkins et al. 2000; Clark et al. 2006), and it is believed that these cytokines may have some therapeutic application in severe malarial anemia.
Increased nitric oxide production in the pathogenesis of anemia of malarial infection
Nitric oxide production increases in any generalized infection particularly so in acute malaria (Clark et al. 1991). Higher levels of nitric oxide produce poor deformability of red cells by inhibiting Na+/K+ ATPase in the red cell membrane and oxidising the membrane lipids through generation of peroxiynitrate. Overactivation of poly-ADP ribose polymerase-1 (PARP-1) by nitric oxide and other proinflammaotry cytokines causes rapid depletion of nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP) from red cells (Clark and Cowden 2003). Hence it can inhibit red cell glycolysis. Membrane-damaged red cells then help up in spleen, and the cytopathic hypoxia caused by high levels of NO also suppresses erythropoiesis (Xie and Wolin 1996; Fink 2001) by mitochondrial damage to erythroid progenitors and early erythroid precursors.
Role of hemozoin pigment in apthogenesis malarial anemia
Increasingly, it is becoming apparent that hemozoin, a product of hemoglobin catabolised by malarial parasites, is biologically active. It catalyzes the formation of free radicals through repeated oxidation–reduction cycles due to its Fe+++ moiety, (Arese and Schwarzwer 1997). A significant association was found between the number of circulating haemozoin-containing monocyctes with severity of anemia and suppression of reticulocyte count by malarial infection (Casals-Pascual et al. 2006). It was also found that the effects of TNF-α and hemozoin in causation of anemia in this infection were synergistic and together cause low interleukin-12 (Luty et al. 2000) and low interleukin-10 production (Nussenblatt et al. 2001). In in vitro studies involving malarial plasma, it was found that amount of hemozoin circulating in the plasma (1–10 μgm/ml) directly and proportionately inhibit erythropoiesis (Casals-Pascual et al. 2006).
Indirect effect of hemozoin through stimulation of proinflammatory cytokines and endoperoxides could conceivably contribute to pathogenesis of anemia in malarial infection. Production of 15-hydroxy-eicosatetraenoic acid (15-HETE) and 4-hydroxy nonenal (4HNE) by hemozion is increased from red cell membrane lipids. These products increase red cell stiffness and shorten red cell life span (Schwarzer et al. 1999; Giribaldi et al. 2004; Skorokhod et al. 2007).
Pitting of malaria parasites and spherocyte formation
Pitting of parasitised red cell in spleen in experimental animal was observed almost 40 years back (Conrad and Denis 1968). Spherocytes were found in high prevalence in peripheral blood in malaria endemic areas, and a detailed study of competing mechanism of whole parasitised red cell removal versus pitting out the parasite from red cell along with some amount of red cell membrane leading to spherocyte formation proved that later mechanism is preferred by the system (Anyona et al. 2006; Kumaratilake et al. 1994). Pitting as one of the major parasite mechanisms is also suggested by high levels of parasite-related antigen on (RESA, pf155) the unaffected spherocytic red cells in malarial infection (Newton et al. 2001; Anyona et al. 2006). Spherocytosis in malarial infection can be caused by several mechanisms.
Immune hemolytic anemia
A proportion of patients develop immune hemolysis in malarial infection (Ghosh et al. 2001; Facer et al. 1979). The reasons for immune hemolysis are multifarious, e.g. antibodies directed to parasitic antigens sticking to red cells, immune complex deposition leading to bystander hemolysis due to parasite antigen or drug antibody complex, or due to oxidative damage and aggregation of red cell anion channel protein and subsequent coating of this denatured protein by naturally occurring autoantibody and subsequent removal by immune system (Turrini et al. 2003). In addition, cytokine dysregulation and increased TNF-α can activate macrophages which, in a hyperactive stage, may even reduce its threshold for amount of antibody coating needed for phagocytosis, i.e. minimally sensitized red cells which otherwise would not have been phagocytosed now are actively phagocytosed due to activation of the monocytes and macrophages.
In addition, anticomplementary defence of red cell membrane, which protects these cells from inadvertent complement-mediated lysis, is also reduced in malarial infection due to loss of complement regulatory protein CD-55 and CD-59 coupled with increased level of immune complexes in malarial infection which makes these cells susceptible to complement-mediated lysis (Stouti et al. 2003).
It is not to be forgotten that antigen from malaria parasites stimulates B cell nonspecifically to produce innumerable autoantibodies, some of which could conceivably be directed towards various red cell antigens (Jhaveri et al. 1997).
In a patient with malarial infection who develops additional immune mechanism for hemolysis, life-threatening anemia can also develop because the infection through various pathways has not only switched off red cell production but also through multiple mechanisms is also destroying red cells peripherally.
Tropical splenomegaly syndrome (hyperactive malarial splenomegaly, hypesplenic malarial syndrome)
In a subset of patients with chronic infection, P. falciparum leads to chronic and intense stimulation of splenic macrophages leading to gross splenomegaly, low levels of parasitaemia and very high levels of IgM in the serum. These patients have a defect in immunoglobulin class switching and a genetic predisposition to develop this condition. Clonal B cell proliferation in this syndrome has also been recognized (Bates et al. 1991). Huge spleen and hyperactive reticulo-endothelial system chronically can cause significant anemia by red cell pooling. A small proportion of these patients develop non-Hodgkin lymphoma, which adds to the existent causes of anemia in this infection as a future consequence.
Parasitised red cell develops special receptors to stick to endothelial cells. This property is seen particularly with P. falciparum infection. Attachment of these parasites can take place through CD-36 ligand (Gamain et al. 2001) or through interaction with endothelial cell chondroition sulphate-like molecule. Cytokine dysregulation could up regulate endothelial adhesion molecules and converts the anti-coagulant endothelium to a procoagulant surface. Hence combination of these two mechanisms may cause intense red cell sequestration in deeper capillaries and disseminated intravascular coagulation (DIC) with hemorrhage. Both these conditions can contribute to acute anemia in P. facliparum infection. A proportion of patients can also develop microangiopathic hemolysis.
Miscellaneous contributory factors
In areas endemic for malaria, there are many contributory factors like nutritional deficiency and other parasitic infestations which have already caused significant anemia or has kept the patient on the precipice; on top of this, when malaria infection takes place, all these multiple causes already preexistent severely aggravate the malarial anemia. Sometimes antimalarial drugs can cause severe gastro-intestinal symptoms or can aggravate anemia by hemolysis or bone marrow suppression (Sulphadoxin + Pyrimethamine, quinine, Primaquin, Chloroquin)
Anemia in malaria could be inconsequential when acute vivax malaria strikes a healthy adult and is immediately treated, or it could be life-threatening in the backdrop of G6PD deficiency with severe acute infection complicated by drug-induced hemolysis. More often, in an endemic area it is often the chronic infection, with miscellaneous other associated causes, that leads to severe anemia. Severe malarial infection causes drop in hemoglobin levels to below 5 gms/dl. Often, children are affected, and the causes are multiple as briefly described in this review. One of the consistent findings in severe malarial anemia is low levels of IL-10 and IL-12, and if we recollect, hemozoin, aptly called a very active “inert” substance, contributes significantly to this anemia, and low IL-10 levels working synergistically with TNF-α and ability of IL-10 has the ability to metabolize hemozion through induction of heme oxygenase 1 (Lee and Chau 2002); it becomes clear why this product ameliorates anemia of malarial infection (Kurtzhals et al. 1997). In Fig 1, an attempt has been made to integrate all the known pathobiological factors that are involved in the pathogenesis of malarial anemia.
One note of caution for those who work in a malaria endemic area: from this brief review, it is clearly seen that malaria can cause generalized immune activation and can give nonspecific positive serological tests like ANA + vity, Direct Coomb’s test positivity in many patients; large number of spherocytes may be confused with hereditary sphereocytosis if the patient is not meticulously looked for malaria infection, and because of staying in endemic area he is partially immune and has little symptoms of malaria. Bone marrow study may be confused with congenital dyserythropoietic anemia in a child with severe malarial anemia. In all these situations in an endemic area, we cannot afford to miss malaria as a cause of many of these mischiefs (Kedar et al. 2002) and should meticulously investigate to rule out malaria infection in a case of inexplicable anemia.