It is helpful to consider the multifaceted nature of the interaction between the host immune system and the parasite. Central to this interaction are cytokines that are released by immunocompetent cells in a highly regulated fashion [¹³]. They participate in the control of all immunologically relevant events, whether they concern either activation, proliferation, and subsequent effector functions of recirculating immunocompetent cells or regulation of cells residing in tissues (for example, resident mononuclear phagocytes and endothelial cells). It has been established that cytokines not only participate in the qualitative (for example, antibody isotype switch) and quantitative regulation of the immune response but also participate in many other complex processes such as hematopoiesis and pregnancy. During the erythrocytic cycle, soluble products of Plasmodium spp. known as malarial toxins direct systemic release of proinflammatory cytokines (for example, tumor necrosis factor-o^∼ (TNF-ix)) which act on many other cellular systems such as endothelium. Equally important are parasite antigens, which stimulate T cells to directly secrete or induce production of cytokines from other cells. Before P. falciparum infection, many individuals have P. falciparum reactive T cells, often at high frequency. Such parasite-reactive T cells have probably arisen as a result of antigenic cross reactivity between environmental organisms and parasite-derived molecules [¹⁴].

Incubation of blood mononuclear cells with parasitized erythrocytes can drive proliferation of these T cells even when the parasitaemia is as low as one parasite per microliter of blood. Because many of these T cells secrete interferon y (IFN-y) and other cytokines and can facilitate production of TNF-o^∼ by monocytes, they have the potential to be involved in disease pathogenesis. Both IFN-^∼ and TNF-cl may play roles in dyserythropoietic anemia, and TNF-a may contribute to cerebral malaria as a result of up-regulation of intercellular adhesion molecule-1 (ICAM-1) in cerebral blood vessel endothelium. With respect to TNF-et production, T cells--whether they express y8 [¹⁵] or oil8 T cell receptors may play as important a role in disease pathogenesis as that postulated for the direct stimulation of mononuclear phagocytes by malarial toxins. It is likely that parasite-dependent activation of T cells and mononuclear phagocytes leads not only to disease but also to killing of parasites. Whether all of the cross-reactive T cells, once activated during the primary infection, will remain functional during a persistent infection or until exposure to re-infection deserves to be studied. The parasite also has other strategies for interacting with the immune system, including the following: antigenic variation; (it) a still undefined, splenic dependent regulation of parasite genes encoding structural proteins and adhesive molecules on the erythrocyte surface that are involved in adherence to endothelium; and low immunogenicity, of conserved parasite peptides that are targets of antibodies able to interfere with parasite survival.

The adherence of parasitized RBCs to other cells is central to the pathophysiology of severe malaria syndromes including cerebral malaria, respiratory failure, multi-organ failure and death [¹⁶,¹⁷]. Parasitized RBCs adhere to the vasculature through a process termed “sequestration” closely mimicking inflammatory leukocyte attachment [¹⁸]. Furthermore, half of infected RBC isolates51 form occlusive intravascular aggregates, which consist not only of infected RBCs bound to each other (“autoagglutinates”) [¹⁹], but also of infected RBCs bound to uninfected RBCs (“homotypic RBC rosettes”) [²⁰] and/or to platelets (“heterotypic RBC rosettes”) [²¹]. Sequestration and rosette formation impair blood flow, causing tissue ischemia and cell death [²²]. Not surprisingly, in-vitro rosetting is more pronounced in parasite strains derived from patients with severe disease, particularly in cases of cerebral malaria [²³–²⁶]. P. falciparum is unique among malaria species in that infected erythrocytes express an adhesive determinant, termed Plasmodium falciparum Erythrocyte Membrane Protein-1 (PfEMP-1). PfEMP-1 is encoded by the parasite genome and is expressed on the outer surface of infected RBCs. PfEMP-1 binds to a variety of target molecules found on RBCs, platelets, and vascular endothelial cells [¹⁶,¹⁷]. The structure of PfEMP-1 includes variable numbers of two types of adhesive binding domains: Duffy binding-like (DBL) regions and cysteine-rich interdomain regions (CIDR). DBL-1α demonstrates lectin-like properties, causing it to bind primarily to cells bearing A and B blood group oligosaccharides, and to other glycosylated targets, such as the glycoprotein CD35 (CR1), and heparan sulfate-like glycosaminoglycan [²⁷,²⁸]. CIDR1, on the other hand, binds principally to CD36 (Platelet Glycoprotein IV), thus targeting platelets and endothelium.

It has been established that parasitized erythrocytes form rosettes more readily with red blood cells of A, B, or AB groups than with cells of blood group O. This parasite-triggered RBC rosette formation is associated with severity of malaria disease [¹⁴]. Rosette formation and autoagglutination are adhesive phenotypes associated with disease severity in P. falciparum malaria [²⁹]. The adherence of parasitized RBCs to other cells is central to the pathophysiology of severe malaria syndromes including cerebral malaria, respiratory failure, multi-organ failure and death [¹⁶,¹⁷].

There is increasing evidence that P. falciparum malaria is influenced by ABO blood group but the extent of association between both is yet to be well defined. P. falciparum, the most dangerous or deadly of the four human malaria parasites, causes about 90% of all malaria deaths in the world today especially in sub-Saharan Africa [⁹,²⁰]. However, no risk associated data for ABO phenotypes and P. falciparum malaria has been reported to date, in Ghana. Thus, this study investigated the hypothesis that ABO blood groups are associated with the severity of P. falciparum infection and the results showed that non-O blood group children are more prone to severe malaria caused by P. falciparum than the group O children.

This study was approved by the Ethics Review Committee of the School of Allied Health Sciences, College of Health Sciences; Korle-Bu. Parental/guardian consent for child participation was sought during the study.

The study was carried out at the Department of Child Health, Korle-Bu Teaching Hospital (KBTH) from May to August 2008. The KBTH is the largest tertiary hospital in Ghana with 17 departments and 1500 beds. One hundred and twenty one (121) children between 3 months and twelve years were sampled.

Patient between 3 months and twelve years old, whose blood film (BF) test was reported as malaria parasites present and thin film showed Plasmodium specie to be falciparum.

Patient below 3 months old or twelve years and above; whose blood film (BF) test was reported either as no malaria parasite present or malaria parasites present and thin film showed Plasmodium specie to be either falciparum or malariae.

Two ml venous blood from the patient was collected into an EDTA-anticoagulated tube (1.2mg of anhydrous dipotassium salt) using a 2ml syringe and needle set. Two drops of blood were placed on a clean glass slide and a thick film and a thin film were prepared on the same slide and allowed to air dry. The thin film was fixed with methanol for one minute. The slides were placed in a staining rack and immersed in a staining trough. Giemsa stain (Wako, Japan) diluted 1 in 10 with phosphate buffer pH 7.2 was used to stain the film for 10 minutes. The stain was washed off the slides using the buffer, pH 7.2. The back of the slides were wiped dry and placed in a draining rack for the film to air-dry.

The parasite density was counted as parasite numbers per microlitre of blood by counting parasites against white blood cells. Using the x100 objective, 200 WBCs were counted systematically, estimating at the same time the numbers of parasites (asexual) in each field covered, with two tally counters.

Calculation = WBC count/µl of patient X Parasites counted against 200 WBCs/200

The above formula was used if after 200 WBCs have been counted, and 10 or more parasites were identified. However, if after 200 WBCs have been counted, and 9 or fewer parasites have been counted, counting continued until 500 WBCs have been counted and the number of parasites was recorded.

Severity of the Plasmodium falciparum malaria disease was determined based on the parasite density calculated as above. Severe parasitaemia: was defined as hyperparasitaemia (> 250,000 parasites/µl or more than 10% of red cells are parasitized); and/or severe anaemia (Haemoglobin concentration of<5g/dl). Moderate parasitaemia: was defined as parasitaemia between 51,000-249,000 parasites/µl and/or Haemoglobin concentration between 5-8g/dl. Mild parasitaemia: was defined as parasitaemia of 1,000-50,000 parasites/µl and/or Haemoglobin concentration>8g/dl.

The spun tube technique [³⁰], a liquid-phase system, was employed for the detection of ABO antigens and antibodies using cell and serum grouping. Four small tubes (75×12 mm) were labeled 1 to 4. Each tube was filled as follows:

• Tube 1-1 volume anti-A serum and 1 volume 5% patient's red cell suspension in saline
• Tube 2-1 volume anti-B serum and 1 volume 5% patient's red cell suspension in saline
• Tube 3-1 volume patient's serum and 1 volume 5% A cells in saline
• Tube 4-1 volume patient's serum and 1 volume 5% B cells in saline

The contents of the tubes were mixed by gently tapping the base of each tube with the finger. The tubes were left at room temperature for 5 minutes and then centrifuged at 150g for a minute. The results were read by gently tapping the base of each tube, looking for agglutination or haemolysis. Red cell reagents (known A, B and O cells) were used to control the antisera (A and B). An autocontrol was also set i.e. patients red cells were added to their own serum to detect the possible presence of autoagglutination. The haemoglobin measurement and leucocyte count were done with an automated haematology analyzer, Sysmex KX-21N. The Sysmex KX-21N employs DC detection method for the leucocyte count and Non-cyanide Sodium Lauryl Sulfate (SLS) method for the haemoglobin measurement.