Review Article. Anemia and Parasitosis Part I: Erythrocytic Parasites. Elmeya H Safar 1 and Magda E Azab 2. Introduction

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1 Parasitologists United Journal (PUJ) Vol. 1, No. 2, July - December, Review Article Elmeya H Safar 1 and Magda E Azab 2 Introduction Anemia associating parasitic infections differs according to the requirements and pathophysiology of the parasites. It stands to reason that the closer the parasite s association with the red blood cells, the more severe the expected anemia. Considering blood parasites, malaria is the most important and well-known infection, followed to a lesser extent by the closely related babesiosis. Anemia is a symptom defined as reduction below normal in number of erythrocytes/ml 3, reduction in the amount of hemoglobin (Hb) which is the oxygen carrying pigment in the blood, or reduction in the volume of packed cells/100 ml blood (1). In severe anemia, the hematocrit (HCT) is less than 20% and Hb is less than 4.4 mmol/l or 7.1 g/dl (2). Anemia develops following blood loss, when red cells are hemolysed prematurely, or when the normal erythroid production of red cells is affected. These mechanisms often overlap with several factors contributing to the anemia. Among the potential causes of increased cell destruction resulting in acquired hemolytic anemia are malaria and babesiosis. Hypersplenism and splenomegaly as in hyper-reactive malaria are also involved in the hemolysis. In these diseases the anemia is normocytic with normal red cell size (MCV fl) and the overall Hb levels are decreased. As a result, the oxygen carrying capacity of the blood is reduced. This is because each Hb molecule is formed of four linked polypeptide (globin) chains each of which is combined with an iron-containing porphyrin pigment called heme. Porphyrin is the oxygen carrying part of the Hb molecule where oxygen combines reversibly to the ferrous (Fe 2+ ) contained in each heme group (3). Anemia and Parasitosis Part I: Erythrocytic Parasites Research Institute of Ophthalmology Giza 1, Parasitology Department, Faculty of Medicine, Ain Shams University, Cairo 2, Egypt Received: October, 2008 Accepted: December, Hence, being an essential component of Hb, iron is needed for its synthesis. In iron deficiency anemia, there is a decrease in the number of red cells in the blood caused by too little iron. The apicomplexan parasites Babesia and Plasmodium, are related yet phylogenetically distinct hemoprotozoa that infect red blood cells and cause severe diseases of major human and veterinary importance. A variety of cellular and molecular interactions are involved in their pathogenicity with striking similarities in their pathophysiology. This review is a summary of the findings of research into cellular adhesive phenomena in malaria and its correlation with babesial-infection. Such information is fundamental in order to learn more about the biology, cellular and molecular mechanisms by which these parasites cause infection and disease. Increased destruction of erythrocytes associated with repeated cycles of growth of Plasmodium and the rare Babesia infections is influenced by both parasite and host factors. Erythrocytic parasites also harm their hosts indirectly, causing physical damage, as a result of the mechanism by which the host s immune system attempts to kill the parasites. Comparison of the cellular and molecular mechanisms that culminate in accumulation of parasitized red blood cells in the microvasculature of humans infected with P. falciparum and animals infected with Babesia spp. is particularly instructive. While such adhesive phenomena have been studied extensively in malaria, they have received relatively little attention in babesiosis. It is increasingly recognized that effects of cytokines released by the host s immune system in response to parasites are responsible for many of the symptoms of the disease. In order to limit the damage

2 62 Safar and Azab caused by an invading parasite, a host s immune system must respond in a balanced and well regulated manner and is therefore a critical determinant of pathogenesis and disease outcomes. A review of the literature produced reports that explain the mechanisms leading to the occurrence of anemia in these two erythrocytic parasitic infections, and the role played by the host s immune response. Keywords: Malaria, Babesiosis, Severe Malarial Anemia, Hemolytic Anemia, Cytoadeherence, Eerythropoiesis, Cytokines. Malaria Malaria is one of the serious and complex health problems that are still mainly confined to poorer tropical areas of Africa, Asia, and Latin America where it is mainly transmitted by Anopheline mosquitoes. Patients may present with a mild febrile illness, or as in P. falciparum infections with a severe life threatening syndrome of a number of distinct but overlapping clinical signs, the most prominent of which are profound anemia with secondary heart failure and cerebral malaria (4). The very rare and dangerous complication of hemoglobinurea or black water fever typically occurs in non-immune residents of malarious areas and is attributed to sudden, extensive, immune lysis of quininesensitized erythrocytes (5), and in some cases following treatment with halofantrine (6). As a result, erythrocyte counts may fall in a few hours to 1x10 12 / litre or less and Hb present in plasma is passed in urine (4). The degree of malarial endemicity varies between countries and even between different areas in the same country. In areas of low endemicity, the disease affects all age groups while in areas of high endemicity, overt disease is greatly reduced despite the harboring of malaria parasites. Over 80% of malarial deaths occur in Africa where 66% of the population is at risk; while less than 15% of the total global deaths from malaria out of 49% exposed to infection, occur in Asia including Eastern Europe (7). One hundred and nine countries were endemic for malaria in 2008, 45 within the WHO African region (8). WHO (9) defined malarial anemia during acute illness as > 200,000 parasites/μl (exceeding 5% of erythrocytes), and/or Hb level < 5.0 g/l, and in mild illness as < 100,000 parasites/μl with Hb level > 5.0 g/l and without signs and symptoms of severe malaria (for a detailed report on the physiology of malarial anemia, refer to English (10) ). Severe malarial anemia (SMA) is one of the major presenting features of falciparum malaria in African children, and is the most frequent presentation in Kenya and in Papua Guinea where majority of deaths associated with it occur within 12 h of admission (11,12). There were an estimated 247 million malaria cases among 3.3 billion people at risk in 2006, causing nearly a million deaths, mostly of children under 5 years. Globally, each year between 75 thousand and 200 thousand infants deaths are attributed to malaria infection in pregnancy, and between 200 thousand and 500 thousand pregnant women develop SMA in Sub-Saharan Africa (13). Primigravidae are the worst affected (14). Infection causes profound anemia after the first trimester where a sudden and catastrophic fall in Hb may occur. In the second trimester, primigravidae are prone to develop SMA that bears little relation to their peripheral parasitemia (15). In a cross-sectional community-based survey of pregnant women in rural Southern Mozambique, the prevalence of clinical malaria was 3%, and that of anemia 59% (16). In endemic areas, the incidence of congenital malaria during the latter half of pregnancy is very low in spite of the concentration of falciparum parasites in maternal blood of the placenta (17). Congenitally infected neonates may have cord blood and peripheral parasitemia that disappears within a day or two. During epidemics, symptoms and/ or signs of malaria infection are more common in neonates born to non-immune mothers. Newborns are protected up to 6 months of age by passive transfer of maternal immunoglobulins and by fetal Hb. Between 6 months and 2 years of age, infection is often the result of repeated untreated or partially treated episodes of uncomplicated malaria. In areas with high transmission, 1-3 years old children present with SMA, while in areas of lower transmission older children present with cerebral malaria (18). High mean asymptomatic parasite density and presence of clinical malaria episode were reported to be major predictors of anemia in children < 24 months (19).

3 Anemia and Parasitosis: Erythrocytic Parasites Because erythrocytes are the target host cells, plasmodium obtains most of its amino acid requirement from digestion of erythrocytes proteins particularly Hb, hence anemia is an inevitable consequence and a major complication of malaria. Researchers have generally agreed that following invasion, the mechanism of Hb pathogenesis by malaria parasites involves: (1) erythrocyte lysis as an integral part of the life cycle of the parasite (2) reduced incorporation of heme (3) splenic removal of both infected and non infected erythrocytes coated with immune complexes (4) autoimmune lysis of coated infected and non infected erythrocytes (5) erythropoietic suppression and dyserythropoiesis (20). Biological features of Plasmodia In areas with natural resistance to malaria, Plasmodium different species have important distinctive characteristic biological features related to the infected erythrocytes (21). Infection by P. vivax is restricted to invasion of reticulocytes bearing Duffy blood group determinants, a feature that explains why red blood cells infected with trophozoites stages are sometimes described as larger than normal. The absence of vivax malaria from areas of West Africa is explained by the high proportion of Duffy negative individuals in the population. A major biological difference is that P. ovale can infect Duffy negative reticulocytes. P. malariae has an apparent preference for old senescent erythrocytes nearing the end of their life span, explaining why infected cells are often described as smaller than normal. Accordingly, there is a natural limit to these infections since reticulocytes constitute less than 2% of the total number of red blood cells, and those suitable for infection with P. malariae are even less. On the other hand, P. falciparum infects mature and young erythrocytes indifferently, consequently resulting in considerable degrees of anemia. Another form of natural resistance operates after merozoites have entered the red cells. Development of P. falciparum is retarded in erythrocytes presenting various hemoglobinopathies including Glucose-6- phosphate dehydrogenase (G6PD), β-thalssemia; mutation of sickle cell hemoglobin (HbS) due to glutamate-to-valine substitution in the sixth position of the β-globin chain (22) ; a variant mutation causing 63 glutamate-to-lysine substitution at position 26 of the β-lobin chain (HbE), fetal Hb (HbF) which is the main oxygen transport protein in the fetus, and hemoglobin C (HbC) which also carries a glutamateto-lysine mutation in the beta globulin chain. The enzyme G6PD normally protects from the effects of oxidative stress in red blood cells. A genetic X- linked deficiency in this enzyme results in increased protection against severe malaria in the population of malarious regions in Tropical Africa. This relative protection occurs in heterozygote individuals whose blood contains both normal and G6PD-deficient red cells where merozoites develop only in the normal cells. While in homozygous females or hemizygous males whose blood contains only G6PD-deficient red blood cells, there is reduced growth of P. falciparum during the initial cycles, and then the parasite is induced to produce its own enzyme and to gradually adapt to growth in the enzyme-deficient cells (23). Thalassemia or sickle cell disease (SS) patients fail to synthesize normal quantities of Hb α2 (thalassemia minor: heterozygous form inherited from one parent) and β2 (thalassemia major: homozygous form inherited from both parents) depending on which chain is synthesized in reduced quantities (24). Heterozygote carriers having one copy of HbS gene project substantial protection against falciparum malaria with lower parasite densities than in normal children; while in homozygous patients, the infection is invariably fatal (25). Thus, sickle-cell anemia is a genetic buffer against malaria that arose as a random mutation and, by chance, provided some resistance to malaria. Several mechanisms have been proposed to explain the impedance of falciparum multiplication to high densities in benign sickle trait heterozygous erythrocytes. One report proposed that the significantly greater sickling rate of parasitized heterozygous erythrocytes promotes their increased removal from the blood by the spleen (26). Another report proposed that parasite development is impaired and in situ death occurs within the sequestrated parasitized heterozygous erythrocytes due to the low-oxygen environment of post capillary venules (27). Moreover, the impaired invasion and development of parasites was reported (28). A genotypic study (29) reported that parasite density is unrelated to the severity of anemia in children with acute malaria. It was found that while homozygosity for the R131 allele

4 64 Safar and Azab protects against high density parasitemia, Fcgamma Rlla-131 polymorphism, to which malaria specific IgG antibodies are bound, does not protect against malaria anemia. Cytoadherence Fairhurst et al. (30) postulated on the fact that HbC protects West African children against P. falciparum. Parasitized heterozygous (HbAC) and homozygous (HbCC) erythrocytes were shown to inhibit the adherence of falciparum-infected parasites to vascular endothelial cells expressing CD36-receptor and intercellular adhesion molecule-1(icam-1), and to impair the rosetting interactions of nonparasitized erythrocytes. The authors explained that HbC protection against malaria may be through reduction of the erythrocyte membrane protein- 1(PfEMP-1) mediated adherence of parasitized erythrocytes. Another study (31) was performed to assess the association between major clinical forms of SMA, HbS, HbC and α-thalassemia. It was found that while the HbS carrier state is negatively associated with all major forms of severe falciparum malaria, carrier state of HbC is negatively associated with cerebral malaria and α is negatively associated with severe anemia. PfEMP-1 is a family of antigens exported by the parasite to the red cell surface. They are composed of large ( kda) molecules concentrated in the electron-dense protrusions or knobs on the surface of parasitized erythrocytes, recognized as Maure r clefts in stained parasites (32), and are the parasite s major cytoadherence ligand and virulence factor (25). PfEMP-1 contains multiple adhesive modules, including the Duffy bindinglike domain and the cysteine-rich interdomain region (CIDR). PfEMP1 specifically attaches to CD36, the main cytoadherence receptor on the surface of endothelial cells and blood monocytes. Interaction between CIDRα and CD36 promotes stable adherence of parasitized cells to endothelial cells. These enable the parasites to avoid clearance from the blood stream by the spleen (33,34). Promotion of cytoadherence by Pfemp-1-CD36 interaction may also be mediated by another receptor, namely thromboplastin, which is a multi-factorial glycoprotein synthesized by endothelial and other adherent cell types and is released on platelet aggregation (4). Cholera et al. (25) probed further the interaction between PfEMP-1 on trophozoites-infected erythrocytes and CD36 on blood monocytes. Their results showed that the significant reduction in binding of parasitized benign heterozygous sickle trait erythrocytes to microvascular endothelial cells and blood monocytes, relative to the binding of normal infected erythrocytes, correlates with a reduced display of PfEMP-1. Erythrocyte invasion and lysis According to species, the rupture of mature schizonts every hours destroys the erythrocytes. P. falciparum has shorter cycles of development that produce large numbers of merozoites thus destroying more erythrocytes than other species. Released merozoites seek new erythrocyte host cells. The process of invasion starts by random contact between the merozoite and the erythrocyte followed by orientation so that its apical complex is directed towards the host cell membrane. Merozoites express a number of antigens at their surface released from organelles such as rhopteries and micronemes. One of these a histidine-rich protein (HRP-2), causes deformation of the physical characteristics of the red cell surface, thus facilitating invagination into the host cell (35). The invaginated membrane then fuses at its external limits to form a parasitopholus vacuole. The erythrocyte s cell membrane within the vacuole disintegrates, bringing the parasite in direct contact with the cell cytoplasm. Sequence of the red cell attachment and invasion takes about 30 seconds (36). An important group of surface antigens (merozoite surface proteins), derived from large molecular weight molecules MSP1 and 2 (or MSA 1 and 2) play a role in interaction with the red cell membrane. In P. falciparum, MSP1 is also referred to as Pf 200 or gp195 (37). The driving invasion of erythrocytes is facilitated by the merozoite s motor systems attributed to an actomysin motor XIV myosin called Pfmyo-A, and to a subpellicular microtubule assemblage called f-mast (38). Reduced incorporation of heme Plasmodium parasites sustain themselves by consuming Hb in the erythrocyte cytosol and a few other amino acids from outside the erythrocyte. It was found that 20% parasitemia results in consumption of 110 g/dl hemoglobin in 48 hours (39). In acute malaria, there is immobilization of iron in

5 Anemia and Parasitosis: Erythrocytic Parasites the macrophages, low serum iron concentration and massive increase in serum of the acute reactive protein ferritin (40). Hemoglobin is broken down within the parasites food vacuoles into an aggregation polymer known as heme, which is the major component of the malarial pigment (hemozoin; Hz), and globin which is further hydrolysed to free amino acid (41). Leucocytes acquire Hz through direct phagocytosis of infected erythrocytes and of free Hz released upon rupture of infected erythrocytes (42). In malaria endemic regions, phagocytosis of Hz by monocytes and neutrophils is considered a better index of disease severity than peripheral parasitemia (43). Up to 75% of the Hb of an infected cell may be degraded by the parasite. As a result, there is decreased incorporation of heme for production of new erythrocytes (21). In SMA caused by P. falciparum, HCT is < 0.15 i.e. Hb concentration is less than 5.0 g/dl in the presence of a parasitemia of > 10 thousand parasites/μl and normocytic blood cells (44). P. falciparum anemia is typically normocytic normochromic with reduced numbers of reticulocytes. When associated with α- and β-thalassemia traits and/or iron deficiency, microcytosis and hypochromia may be present (45,46). Splenic removal of both infected and non infected erythrocytes coated with immune complexes Altered formation of erythrocytes: While normally erythrocytes undergo considerable deformation to traverse the capillary bed, the surface and shape of erythrocytes harboring trophozoites and schizonts is visibly changed. Reduced deformability of falciparum infected erythrocytes is directly proportional to the maturity of the intracellular parasite, where the larger the parasite, the more rigid the red cell becomes (47). Another alteration is due to the presence of PfEMP-1 knobs found on the surface of infected erythrocytes. Accordingly, falciparum-infected erythrocytes become distended and lose their ability to change form in order to squeeze through fenestrations of endothelial cell walls, and become adherent to capillaries in the spleen and tissues of other organs. In addition, oxidation of uninfected erythrocyte membranes and exposure to proinflammatory cytokines have been implicated in causing their reduced deformability and consequent clearance by the spleen (48,49) (for more factors accounting for reduced deformability of RBC, refer to WHO (4) ). Sludging of blood flow 65 occurs due to sticking of parasitized erythrocytes together and inability to pass through the capillary bed. PfEMP-1 and ring surface protein (RSP-2) bind the infected erythrocytes to ligands on the surface of endothelial cells forming focal junctions with capillary endothelium. This cytoadherence is responsible for the sequestration of the parasiteinfected erythrocyte (44). Other host cell molecules such as CD36, thrombospondin (TSP) and ICAM-1 may function as endothelial cell surface receptors for falciparum-infected erythrocytes. In P. vivax and ovale, there are small depressions or caveolae on the infected erythrocyte surface connected by a network of small vesicles and clefts which readily appear with Romanowsky stain as reddish cytoplasmic stippling or Schuffner s dots. P. vivax does not cytoadhere in the deep capillaries of inner organs and also P. malariae infected red blood cells do not exhibit cytoadherence and therefore no sequestration. It was postulated that P. vivax infected red cells specifically adhere to barrier cells in the human spleen allowing the parasite to escape spleenclearance. Merozoites are consequently released in an environment where reticulocytes are stored before release into the circulation to compensate for the anemia associated with vivax malaria (50). In P. falciparum only, the early ring stages are present in the circulation and later, developmental stages evade the spleen by cytoadherence and sequestration in other organs including heart, liver and brain (51). While the peripheral circulation shows low parasitemia, cerebral capillaries contain a high proportion of enlarged erythrocytes predominantly parasitized with late trophozoite and schizont forms (52). Infected erythrocytes sequester in the placenta, where rich capillaries and weak immune responses create a hospitable environment for the parasite. They accumulate in the placenta through surface-adhesion to maternal receptor molecules such as chondroitin sulphate A (53). Acute placental insufficiency can have harmful consequences for the fetus interfering with its supply of oxygen and nutrients and increasing the risk of premature delivery. Autoimmune lysis of coated infected and non infected erythrocytes: Enhancement of phagocytic activity, due to lymphoid and macrophage hyperplasia, results in splenic enlargement. With repeated attacks

6 66 Safar and Azab of malaria (especially in children), the anemia is disproportional to numbers of parasitized red cells suggesting associated sensitization and destruction of non infected red blood cells. It has been estimated that 10 times as many uninfected erythrocytes are removed from the circulation for each infected erythrocyte (54). Sensitization mechanism may be through production of auto-antibodies to the red cells, or through binding of soluble malarial antigens or of circulating antigen-immunoglobulin complexes to the cell surface. These parasite products include the P. falciparum RSP-2 which besides mediating adhesion of infected RBCs (55,56) is also deposited on uninfected RBCs promoting their opsonization and leading to SMA (57). Parasite-specific antibodies, IgM, IgG1, IgG2, IgG3, and IgG4 are produced in response to the parasite antigens expressed on surfaces of erythrocytes. The presence of antibody and immune complexes on the surface of the erythrocytes stimulates antibody dependent cellular cytoxicity (ADCC), complement-mediated hemolysis and erythrophagocytosis. Most of malaria infected patients show positive direct Coombs test due to absorption of IgG, immune complexes, and complement (58). Suppressed erythropoeisis and dyserythropoiesis Severe malaria is associated with bone marrow abnormalities including dyserythropoiesis, ineffective erythropoiesis and reduced proliferation of erythroid colonies (59). Moreover, RSP-2 present on the surface of erythroblasts in the bone marrow lead to clearance or damage of circulating or developing erythroid cells contributing further to SMA (44). Dyserythropoiesis was observed only in chronic anemia. Children with chronic anemia (parasitemia < 1%) had higher levels of erythroid hyperplasia and dyserythropoiesis than those with acute anemia who showed normal erythroblastosis, despite an increase in bone marrow cellularity indicating suppression of erythroid response. Disruption of red cell production by the bone marrow coincided with reduced reticulocytosis (60,61) relative to the degree of anemia in children with malarial anemia, even in the presence of elevated levels of erythropoietin (62). Erythropoietin production is enhanced in response to fall of Hb levels and the subsequent reduction of oxygen tension and has been shown to be appropriately raised in malarial anemia in African children (63). However, other studies in adults from Thailand and Sudan suggested that though raised, the erythropoietin concentration proved inappropriate for the degree of anemia (64) (for regulation of hematopoietic activity, refer to Lamikarna et al. 44) ). Thrombocytopenia is among the bone marrow abnormalities and although extremely common in severe falciparum malaria, it is not related to other measures of coagulation as prothrombin time and partial thromboplastin time, or to plasma fibrinogen concentration, and in most cases is accompanied by bleeding. It was reported (65) that in a small proportion of Thai adults, about 10% with acute malaria may be complicated by disseminated intravascular coagulation (DIC) as a result of the release of thromboplastin during massive hemolysis, toxic destruction of endothelium and the activation of complement. Anemia and reticulocytes suppression were also found to be associated with Hz-containing monocytes and plasma Hz independent of the level of circulating cytokines, including tumor necrosis factor (TNFα) (66). The authors showed that not only isolated Hz, but also dilapidated Hz, inhibits erythroid development in vitro in the absence of TNF-α which when added to cultures was found to synergize with Hz to inhibit erythropoiesis. Hz scavenged by monocytes may also be involved in impairment of erythropoiesis by reducing macrophage oxidative burst activity preventing up-regulation of activation markers (67,68) and stimulation of biologically active endoperoxides from the monocytes (69,70). In addition Hz has been shown to stimulate the innate proinflammatory response (71). From in vivo studies in infants and children residing in a holoendemic P. falciparum transmission area, and in vitro cultured peripheral blood mononuclear cells from malaria-naïve individuals, it was demonstrated that migration inhibition factor (MIF) is suppressed in children with SMA and that monocyte-acquired Hz plays a role in promoting SMA and decreasing MIF production (72). It was concluded that acquisition of Hz by monocytes leads to suppression of MIF production and enhanced severity of anemia in childhood malaria. Macrophage support of terminal differentiation of erythroblastic islands in the bone marrow may also be disrupted by the engulfed

7 Anemia and Parasitosis: Erythrocytic Parasites Hz (44). The quantity of Hz in erythroid precursors and macrophages were found to be abnormally associated with the proportion of erythroid cells in bone marrow sections from children who had died of severe malaria (66). Cytokines Whether the immunological response to infection is protective or detrimental to the host is determined by the balance between pro-inflammatory and antiinflammatory cytokines production. Peripheral blood MIF is a pleiotropic cytokine released by monocytes/macrophages T cells, and cells of the anterior pituitary gland (73-75). This factor, MIF regulates innate immunity and pro-inflammatory cytokine production of IL-12, TNF-α (76) and nitrous oxide (NO) (77) which are considered important mediators of innate immunity to malaria (78,79). Migration inhibition factor is also important in adaptive immune responses through promotion of T cell and B cell activation and proliferation, and antibody production (74). Produced by activated T cells and macrophages, MIF acts through inhibition of the anti-inflammatory activity of glucocorticoids (80). It was reported that MIF was elevated in patients with more severe anemia, where it conveys protection by promoting a more efficient control of the initial phase of parasitemia (81). Elevated levels of MIF protein were also observed in blood vessel walls of Malawian children with cerebral malaria (80) and in intervillous blood mononuclear cells from women with placental malaria (82). Recent investigations on circulating MIF in children were the first to report that peripheral blood MIF concentrations and peripheral blood mononuclear cell MIF mrna expression were reduced in Gabonese children with mild to moderate forms of malarial anemia and hyperparasitemia (83). In another recent report, it was shown that children with prior mild malaria had higher plasma MIF levels and peripheral blood mononuclear cell MIF transcript levels than children with an identical number of previous episodes of severe malaria, suggesting the importance of increased basal MIF production in generating immune responses that protect against the development of severe malaria (84). The pro-inflammatory Th1 cytokines as TNF-α and interferon (IFN-γ), and functionally related 67 Th2 cytokines as the interleukins (IL-10,-4) are produced as a normal part of the host response to infection. Stimulated macrophages in the spleen and cytokines especially TNF-α, INF-γ and IL-10 destroy both parasitized and unparasitized red blood cells and depress erythropoiesis, further contributing to anemia (85). In SMA, the immunologic expression of TNF-α, a circulating inhibitor of erythropoiesis, functionally antagonizes the action of erythropoietin. The red cell precursors, or normoblasts, show cytoplasmic vacuolation, basophilic stippling, intracytoplasmic bridges, nuclear fragmentation (karyorrhexis), incomplete and unequal nuclear division, and multinuclearity (86). Cytokines are also responsible for the increased uptake and intracellular storage of iron by macrophages, where it is used for production and function of reactive oxygen intermediates (ROI), which destroy infected erythrocytes (87) ; and NO which is the main effector molecule of antimicrobial toxicity in macrophages, takes part in the killing of the malaria parasite (88). Due to its uptake by macrophages, iron is decreased in the circulation resulting in decrease of parasite growth and impairment of erythrocyte production in the bone marrow (89). The severity of anemia appears to depend on levels of TNF-α relative to its anti-inflammatory regulator IL-10, where SMA in children was found to be associated with low ratio of plasma IL-10/TNF-α (90). In another study, while the highest concentrations of TNF-α were found in children with malaria anemia, IL-10 levels were highest in children with highdensity uncomplicated malaria (91). The mean ratio of IL-10:TNF-α was significantly higher in children with mild and high-density parasitemia than in children with malaria anemia. Thus, higher levels of IL-10 and transforming growth factor (TGF-β) over TNF-α and IL-10 may prevent development of malaria anemia by controlling their excessive inflammatory activities. Suggested roles for IL- 12 are the enhancement of the development of protective immunity, and alleviation of malariainduced anemia by increasing the number of erythroid precursors and enhancing the expansion of committed erythroid progenitors (92). In severe malaria, the role of IL-12 is controversial with reports observing moderately increased levels (93) or no significant increase or decreased level (94). This

8 68 Safar and Azab was also associated with increase and decrease in levels of anti-inflammatory cytokines such as IL-10 and TGF-β (reviewed by Sri-Hidajati, 2005) (89). The multifaceted effects of TNF-α in the pathophysiology of malaria is summarized by Hommel and Giles (21) where Plasmodium schizonts produce malaria endototoxin which stimulates macrophages to release TNF-α, and produce parasite antigens which stimulate CD4+T cells to release γ-ifn, GM-CSF, IL-3 and T-cell γδ, and these in turn also stimulate macrophages to release TNF. Low doses of TNF are protective (anti-inflammatory) causing destruction of intracellular parasites. High doses of TNF have pathological consequences (pro-inflammatory) causing anemia (abnormal erythropoiesis and phagocytosis), increased cytoadeherence coma (cerebral malaria), and other symptoms (fever, headache, thrombocytopenia, vomiting, nausea). The main processes involved in parasite damage and host responses resulting in disease and mortality are outlined by Gray (95) where rupture of erythrocytes and release of parasites toxins results in a) increased loss of blood cells and consequent anemia and b) release of TNF and other cytokines which promote (i) increased macrophage activity and consequent increased loss of blood cells and anemia, (ii) reduced erythropoiesis leading to anemia, (iii) fever and other symptoms, (iv) hypoglycemia and metabolic acidosis, (v) cerebral malaria and other severe complications. Metabolic activities of the parasite in turn result in hypoglycemia and metabolic acidosis; and reduced deformability and sequestration of red blood cells lead to blocked capillaries and consequent hypoxia due to blocked capillaries that in turn cause (i) hypoglycemia and metabolic acidosis (ii) cerebral malaria and other severe complications. Babesiosis Babesiosis otherwise known as piroplasmosis is due to infection with Babesia species. These are intraerythrocytic parasites that are essentially parasites of wild and domestic animals. Infection occurs by the bite of species of the hard tick Ixodesi. Numerous types of mammals serve as hosts for these parasites. Babesia causes malaria-like symptoms and hemolytic anemia and its pathology like that of malaria is caused by asexually multiplying intraerythrocytic forms. The first documented human case reported was in 1957 in a splenoectomized resident of former Yugoslavia who died of anemia, fever hemoglobinuria and renal failure. Intra-erythrocytic parasites detected were tentatively described as those of B. bovis of cattle (96). Nearly a decade later in 1969, the second diagnosed case was in an elderly non splenoectomized resident of Nantucket island, USA due to infection with the rodent piroplasm B. microti (97), which is the commonest pathogen causing infection in humans who have no predisposing immunocompromising factors, while producing a self-limiting infection and resistance to reinfection in mice (98). The first case of human babesiosis reported from Germany (99) was in a 6-year-old splenoectomized patient with relapse of nodular lymphocyte Hodgkin s lymphoma complicated by anemia and hemoglobinuria. In 1982, Ortiz and Eagle reported ocular findings in a 34-yearold splenoectomized woman in whom ophthalmic examination showed occasional splinter hemorrhage and bilaterally and centered hemorrhages above the left optic disc; the patient s hospital course was marked by persistent hemolytic anemia (100). Since then, hundreds of cases have been reported from the USA, Taiwan and South Africa (101). In 1991, another canine Babesia gibsoni-like piroplasm (WA1) was identified in a human case from Washington, proved later to be genetically distinct from B. microti and B. divergens (102). Other infections were attributed to B. duncani isolates WA1 and CA5, and B. duncani type piroplasms WA2 and CA6 that proved to be genetically distinct from divergens and microti spp. and related to the canine B. conradae. WA1 strains were previously associated with flulike symptoms in humans and in some cases with disseminated intravascular coagulation, pulmonary edema, and renal insufficiency (103,104). Babesial infection in newborns is rare. A case of transfusionassociated neonatal babesiosis was described in a preterm infant with jaundice, hepatosplenomegaly, anemia and conjugated hyperbilirubinemia (105). The authors reviewed 9 other cases, including 6 that were transfusion-associated, 2 congenital and 2 tick transmitted. Merozoites inoculated with the tick s saliva enter lymphocytes and undergo schizogony the progeny of which infects erythrocytes (106). Babesia spp.

9 Anemia and Parasitosis: Erythrocytic Parasites resemble Plasmodium in that parasites enter red cells without disrupting the cell membrane, and differ in that they lack an exo-erythrocytic cycle so the liver is usually not affected. Different species vary in size where B. microti measures 2 X 1.5 µm, B. divergens measures 4 X 1.5 µm, and B. bovis measures 2.4 X 1.5 µm. The parasites form round, oval or pyriform bodies (piroplasms) which together with their peripheral location in the erythrocytes may confuse them with ring forms of P. falciparum. Reproduction is asexual by budding into 2-4 merozoites forming a pathognomonic quadruplet form that resembles a Maltese Cross (107). Unlike in Plasmodium, this form of division is asynchronous, and infected erythrocytes are not enlarged and do not contain stippling. As parasites leave the erythrocyte, the membrane is damaged. Merozoites go on to invade other erythrocytes by a process similar to that described for Plasmodium via multiple adhesive interactions of parasite ligands secreted from the apical organelles. Identified sialic acid residues, protease-sensitive proteins, or sulphated glycosaminoglycans, are suspected as the host receptors of erythrocyte invasion by Babesia parasites (108). By ultrastructural studies of B. microti, an apparently adhesive surface coat surrounding extracellular merozoites is shedded during the entry process (109). Within the erythrocytes, the babesial trophozoites feed on Hb in a different manner from Plasmodium, probably by taking in red cell stroma by pinocytosis. A comparison of the composition of amino acids produced by B. rodhaini and P. berghei indicated that Hb was the main source for both parasites (110), and proteolytic enzymes capable of Hb digestion were detected in bovine Babesia (111). Although digestion of Hb by Babesia parasites does not produce Hz granules as with malaria trophozoites, in a murine model infected with WA1 strain, histopathology revealed deposition of an ironnegative pigment in multiple organs (102). Parasiteinduced changes in the host cell membrane by B. bovis infections facilitate the entry of glucose (112) and nucleosides (113), apparently for parasite metabolism. Infected erythrocytes also show change in shape and size (114) that is not consistent with Plasmodium spp., increased sedimentation rate (115), changes in osmotic fragility (116), increased susceptibility to 69 perforation by electric pulses (117), and nucleoside permeation sites induced in the cell membrane (113). Other changes contributing to increased cell membrane rigidity and cytoadherence characteristic of B. bovis-infected erythrocytes include increased malonyl-dialdehyde and total lipid, decreased vitamin E, sialic acid, and adenosine triphosphate, and the presence of phosphatidyl serine on the outer surface of the cell membrane (118,119). By scanning electron microscopy, membranes of human erythrocytes infected by B. microti appeared damaged. Evident protrusions, inclusions and perforations suggested that red cell destructions are parasite mediated (120). A study of sequestration of B. bovis-infected red blood cells showed that bovine red blood cells become rigid and adhere to vascular endothelial cells (121). These alterations are accompanied by the appearance of ridge-like structures on the red cell surfaces that are analogous to, but morphologically and biochemically different from the knob-like structures on the surface of human red cells infected with P. falciparum, and therefore are not visibly projected on the surface of infected erythrocytes. As in human infection with P. falciparum, when brain tissue from B. bovisinfected cattle was examined, cerebral capillaries were packed with infected erythrocytes that formed focal attachments with cerebral endothelial cells at the site of these knob-like projections (122). In animals, Babesia canis rossi, Babesia bigemina and Babesia bovis cause particularly severe forms of the disease that include a severe hemolytic anemia, with positive erythrocyte-in-saline-agglutination test indicating an immune mediated component to the hemolysis. This mechanism of hemolytic anemia in acute cases has been described (123) as secondary immune modulated due to the development of IgG and/or IgM antibodies that attach to the red cell membrane and activation of complement system. Assays for complement in rats infected with Babesia rodhaini (124) revealed depletion of C2, C3, C4, C5, and whole complement in the course of infection. No evidence of depletion in the alternative (properdin) pathway was found. These findings are consistent with the conclusion that the classical complement pathway is activated during the course of babesial infection. In acute B. microti infections, C3 and C4 were reported to be suppressed (125). Complement

10 70 Safar and Azab proteins bind to the red blood cell and C1, a serine protease from the liver, enters the complement cascade and generates a membrane attack complex that attaches to the red cell and causes holes to be punched in its membrane. This allows the influx of water and electrolytes, cell swelling and lysis. Ensuing partial erythrophagocytosis by macrophages in the spleen produces spherocytes. Super oxide anions were found to be increased in B. gibsoni parasitized-erythrocytes suggesting oxidative damage of erythrocytes due to lipid peroxidation by the parasites (126). During acute B. bovis infection in cattle, hemolytic complement activity (C H 50) levels remained low for about 2 weeks after parasitemia had declined (127). Severe anemia in spite of a low parasitemia in B. gibsoni infected dogs was shown to be due to the high degree of IgG-bound erythrocyte loss, via immunological membrane oxidative damage and phagocytosis of erythrocytes by activated macrophages (128). Most human infections with B. microti are asymptomatic (129), though it also results in hemolytic anemia which may last for several days to a few months (130). Acute illness in humans appears suddenly. Manifestations are of a malarialike illness including asynchronous fever, chills, sweating, myalgia, fatigue, hepatosplenomegaly and anemia. A parasitemia of 1-2% was reported in mild cases increasing to 85% proving to be severe or even fatal in European immunocompromised patients or those who were splenoectomized prior to B. divergens infection(120). Intravascular and extravascular hemolysis develops as the parasitemia rises, resulting in profound anemia. Fulminating parasitemia results in persistant non-periodic high fever (40-41 C), pathognomonic hemolytic anaemia, thrombocytopenia, atypical lymphocyte formation, hemoglobinuria and renal failure due to induced intravascular hemolysis. In severely anemic cases, Hb concentration fell to < 10 g/dl (131,132). Rarely, infection may be associated with marked pancytopenia, bone marrow hemophagocytosis and marrow histiocytosis (133). Reticulocytosis occurs in response to the hemolytic anemia (134). Among those severely affected, alterations in red blood cell membranes cause decreased conformability, increased red cell adherence, and disseminated intravascular coagulation predisposing to development of acute respiratory distress syndrome (ARDS) (131,132,135). Increased levels of IgM, IgG, and CIq binding and decreased C3 and C4 levels were recorded for acute phase sera from humans with B. microti infection. As in cattle, CH50 levels were below normal in humans tested months after acute B. microti infections (125). In acute babesiosis splenomegaly is due to removal of damaged infected erythrocytes, influx and proliferation of lymphocytes, phagocytic monocytes and natural killer (NK) cells during the developing infection (136) and of compensatory erythropoeisis during recovery (137). In the spleens of B. microti, B cells proliferate more than T cells, and within the latter population, there is a greater increase in helper (L3T4) than suppressor/cytotoxic (Ly-2) cells, as well as an increase in the null-cell component which may contain (NK) cells (138). The possible role for NK cells in mediating protection is demonstrated by its activity during peak parasitemia and the recovery phase (139). Evidence of NK cell activity has also been obtained from studies on human babesiosis. A cell population with characteristics of NK cells was found to be significantly elevated in patients with acute babesiosis (125). A recent case report also showed a marked increase in NK cells during the acute phase of the infection (140). Extreme NK cell activity during peak parasitemia and recovery phases of B. microti infections was associated with increased interferon production (141), supporting the suggestion that interferon mediates NK cells activity (142). The relatively greater severity of human babesiosis in patients lacking a spleen also highlights its important role in limiting parasitemia. In particular, early in infection parasitized and otherwise altered erythrocytes are trapped and phagocytosed by macrophages in the spleen which is the organ responsible for both innate responses and adaptive immune responses involving splenic NK cells and type-1 regulating cytokines IL-12 and IFN-γ (143). It is interesting to note that the production of IFN-γ by CD4+ T cells was found to be at least partially responsible for the resolution of parasitemia after primary infection (144). There is some evidence suggesting that IFN-γ could be directly toxic to intracellular parasites, including those that are intra-erythrocytic (reviewed by Yabrov) (145).

11 Anemia and Parasitosis: Erythrocytic Parasites Also macrophage stimulation has been found to inhibit parasite growth in infected mice through the production of NO (146). Another macrophage soluble mediator, TNF-α, has as well been proposed to mediate parasite death in babesiosis (147). There is also evidence that supports a role for reactive oxygen species (ROSs) in the intraerythrocytic killing of B. bovis (148). Measurement of lymphocyte subpopulations, serum levels of cytokines and adhesion molecules in a case of acute babesiosis showed that the ratio CD4+:CD8+ lymphocytes was reduced versus an increase in NK cells (140). Moreover, as an acute phase response, serum levels of TNF-α, IF-γ, IL-2, IL-6, E-selectin, vascular cell adhesion molecule- 1 and intercellular adhesion molecule-1 were highly elevated. Increase in these biological effects contribute to the associated tissue injury brought about by TNF-α mediated leukocyte adhesion to activated endothelium that is detrimental to the host. TNF enhances procoagulant activity of endothelial cells thus contributing to cytoadherence and coagulation disturbances (149). In experimental WA1 babesial infection, TNF-α mediated hypertrophy and upregualtion of intracellular adhesion molecule- 1 resulted in pulmonary edema, infiltration and adhesion of mononuclear cells to pulmonary veins and endothelial cells activation, whereas resolving B. microti infections were characterized by increased IL-10 and IL-4. In addition, it was further suggested that while CD8+ T cells may contribute to the WA1-associated disease, CD4+ T cells and IFN-γ participate in parasite elimination (for details explaining the role of TNF-α and other cytokines, refer to Hemmer et al. (150) ). In conclusion, damage to erythrocytes is the main pathologic effect of babesiosis and thus variability and severity of host responses depend on numbers of destroyed or altered erythrocytes and their distribution within the microcirculation. Acute hemolytic anemia is induced by those species of Babesia that have no tendency to accumulate in capillaries as in acute B. bigemina, B. equi and B. rodhaini infections. Accumulating species as B. bovis and B. canis cause coagulation disturbances which have also been reported with B. bigemina (151). While hemolytic anemia in acute babesial cases 71 is secondary immune modulated due to the development of IgG and/or IgM antibodies that attach to the red cell membrane with activation of complement system, there is evidence to suggest that protection against babesial infections could be mediated through nonspecific components of the immune response, the so-called innate immunity. Cells of the innate immune system are responsible for controlling the growth rate of the parasite and therefore the extent of parasitemia. Several specific molecules involved in innate immunity have been elucidated in the past several years. Specifically, NK cells and macrophages have been implicated in anti-babesial activity. In the absence of macrophages and NK cells, a higher parasitemia develops in a shorter period of time. The inhibition is most likely accomplished by the production of soluble factors: IFN-γ by NK cells and TNF-α, NO, and ROSs by macrophages. However, it is unclear how these molecules can interfere with the development of the parasite inside the erythrocyte. In addition, T cells can be considered vital in developing resistance to Babesia and CD4+ T helper cells are the subpopulation mainly responsible. Patients with acute infection have an increase in T-suppressor lymphocytes, T-cytotoxic lymphocytes, or both and decreased responses to lymphocyte mitogens with a polyclonal hypergammaglobulinemia. References 1. World Health Organization. Report of a WHO Group of Experts on Nutritonal Anemia. Technical report series No Geneva, WHO; World Health Organization. Malaria Action Programme, Severe and Complicated Malaria. Trans Roy Soc Trop Med Hyg; (Suppl):P Cheesbrough M. (Ed.) District Laboratory Practice in Tropical Countries, Part 2. Cambridge University Press etc; 2000, p: World Health Organization. Severe falciparum malaria. World Health Organization, Communicable Diseases Cluster. Trans R Soc Trop Med Hyg; 2000, 94 (Suppl 1). 5. Bruce-Chwatt LJ. Quinine and the mystery of black-water fever. Acta Leidensia; 1987, 55: Mojon M, Wallon M, Gravey A, et al. Intravascular hemolysis following halofantrine intake. Trans Roy Soc Trop Med Hyg; 1994, 88: World Malaria Report. wmr2005.