PREVALENCE AND SEVERITY OF HYPOGLYCAEMIA AND LACTIC ACIDOSIS IN CHILDREN DIAGNOSED WITH Plasmodium falciparum MALARIA

ABSTRACT

The prevalence and severity of hypoglycaemia and lactic acidosis in Nigerian children diagnosed with Plasmodium falciparum malaria were determined in 100 outpatient children aged 3-144 months (12 years). The children were grouped into 2 categories: 3-59 month old and 60-144 month old. The results obtained indicated that out of the 100 children recruited into this study, seventy-five (75%) were infected while twenty-five (25%) were uninfected with Plasmodium falciparum malaria. On the basis of age group, higher incidence of malaria was recorded in children under 5 years of age with prevalence rate of 85.3%, while those above 5 years had low prevalence rate of 14.7%. The mean blood glucose concentration of malaria-infected children below 5 years (3.80 ± 0.73 mmol/l) was lower than that of malaria-infected children above 5 years (4.21 ± 1.34 mmol/l); however, the difference was not significant (p>0.05). Comparatively, the mean glucose concentrations of the corresponding uninfected subjects were 4.10 ± 0.87 and 4.26 ± 0.51 mmol/l respectively. The mean blood lactate concentration of children below 5 years of age (2.59 ± 1.63 mmol/l ) was significantly (p<0.05) higher than those above 5 years (2.30 ± 1.75 mmol/l). The mean values for both groups were also above the normal range of 1.0 – 2.0 mmol/l while the mean haemoglobin concentration of malaria-infected children below 5 years (16.11 ± 2.24 g/dl) was slightly lower than that of malaria-infected children above 5 years (16.36 ± 2.64g/dl) though not significant (p> 0.05). The prevalence rates of 14.7% were recorded for both hypoglycaemia and lactic acidosis in malaria-infected subjects while 16.0% was recorded for anaemia. There was no significant correlation between blood lactate concentration and blood glucose concentration (r= 0.032, p=0.751) but there was significant positive correlation between haemoglobin level and glucose concentration (r=0.401, p=0.0001). The results suggest that the risk of hypoglycaemia, lactic acidosis and anaemia is higher in younger children, particularly among those below five years of age and also confirmed the knowledge that malaria is a major cause of hospital visits by children.

CHAPTER ONE

                                    

                                             INTRODUCTION

 

Plasmodium falciparum is the most common cause of severe and life-threatening malaria, which causes over 2 million deaths every year (Bruneel et al., 2003; Njuguna and Newton, 2004). In Africa, a vast majority of these deaths occur in children under five years of age (WHO, 2012). Lactic acidosis complicates 35% of severe childhood malaria (Krishna et al., 1994) and hypoglycaemia is present in 20% of children with cerebral malaria (Newton and Krishna, 1998). Both acidosis and hypoglycaemia commonly coexist but each is considered separately as a cause of fatality in children and adults due to severe complicated malaria. Hypoglycaemia is known to be an independent risk factor for death in both severe malaria (Gray et al., 1985; Molyneux et al., 1989) and other severe childhood infections in the tropics (Kawo et al., 1990). Despite its importance, its pathogenesis is not well understood (English et al., 1998). Hypoglycaemia is associated with a poor prognosis in severe malaria (krishna et al, 1994).

In African children with malaria, impairment in hepatic gluconeogenesis in the presence of adequate levels of precursors (glycerol) has been considered the most likely mechanism (White et al., 1987). Irreversible coma may quickly develop if the condition is not effectively treated. Hyperlactataemia is often associated with a poor outcome in severe malaria in African children (Krishna et al, 1994). The pathophysiology of metabolic acidosis is complex. The direct contribution of P. falciparum to the final lactate concentration, through anaerobic glycolysis in the parasite itself, is likely to be small (Vander et al., 1990). More significantly, an inadequate supply of oxygen to tissues may follow from severe anaemia and provoke a metabolic shift within host cells to anaerobic glucose metabolism and increased lactic acid production. In addition, the flow of blood through the microcirculation may be impeded by adherence of infected erythrocytes to the endothelium of post-capillary venules and/or increased rigidity of uninfected cells (Dondrop et al., 1997). Lactate may not in itself be sufficient to cause acidaemia but the inhibition of oxidative metabolism in the context of an ongoing inflammatory response will cause protons (H⁺) to accumulate and eventually lead to metabolic acidosis (English et al.,1997). These pathophysiological pathways suggest that the syndrome of lactic acidosis may be associated with the total parasite burden during acute infection.

Acute malaria is estimated to cause 225 million cases of ill health per year, resulting in over one million deaths per year, most of which occur in sub-Saharan Africa (World Malaria Report, 2010; Murray et al., 2012). Malaria is particularly virulent among children, constituting one of the principal causes of child morbidity as well as mortality in sub-Saharan Africa (WHO, 2000). Exposure to the malaria parasite not only results in bouts of high fevers among children, but also increases the risk of malnutrition and anaemia among children under five (Ehrhardt et al., 2006).

 

• Malaria

Malaria is world's most widespread infection. According to the World Malaria Report 2011, malaria is prevalent in 106 countries of the tropical and semitropical world, with 35 countries in central Africa bearing the highest burden of cases and deaths (World Malaria Report, 2011). Compared to a century earlier, the area of malaria risk has reduced from 53% to 27% of the Earth’s land surface and the number of countries exposed to some level of malaria risk has fallen from 140 to 106 (Hay et al., 2004; Uk Aid, 2010; World Malaria Report, 2011). In 2007, 2.37 billion people were estimated as being at risk of Plasmodium falciparum malaria worldwide, with 26% located in the World Health Organization Regional Office for Africa (WHO/AFRO) region compared to 62% in the combined South-East Asia and Western Pacific Regional Offices (SEARO/WPRO) regions (Uk Aid, 2010). Of this total population at risk, about 42% or almost 1 billion people lived under extremely low malaria risk (Uk Aid, 2010).

Of the five Plasmodia species that infect human beings (P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi), P. falciparum and P. vivax cause the significant majority of malaria infections. P. falciparum, which causes most of the severe cases and deaths, is generally found in tropical regions, such as sub-Saharan Africa and Southeast Asia, as well as in the Western Pacific and in countries sharing the Amazon rainforest. P. vivax is common in most of Asia (especially Southeast Asia) and the Eastern Mediterranean, and in most endemic countries of the Americas.

 

Estimates of the annual incidence of malaria vary widely. According to the estimates of The World Malaria Report, 2011, there were 216 million episodes of malaria in 2010, of which approximately 81%, or 174 million cases, were in the African Region (World Malaria Report, 2011), about 91% being due to P. falciparum (World Malaria Report, 2011). But the actual number of cases may be much more and the number of confirmed cases reported by national malaria control programmes was only 11% of the estimated number of cases (World Malaria Report, 2011). Hay et al have estimated the number of clinical cases of P.falciparum malaria in 2007 at 451 million (95% credible interval 349-552) (Hay et al., 2010; Uk Aid, 2010). According to the estimates of The World Malaria Report, 2011, the vast majority of cases (81%) were in the African Region followed by the South-East Asia (13%) and Eastern Mediterranean Regions (5%) (World Malaria Report, 2011). Nineteen countries in Africa – Rwanda, Angola, Zambia, Guinea, Chad, Mali, Malawi, Cameroon, Niger, Burkina Faso, Côte d'Ivoire, Ghana, Mozambique, Uganda, Kenya, United Republic of Tanzania, Ethiopia, Democratic Republic of the Congo (DRC) and Nigeria – accounted for 90% of all WHO estimated cases in 2006 (Uk Aid, 2010). Hay et al reported that more than half of all estimated P. falciparum clinical cases occurred in Nigeria, the DRC, Myanmar (Burma) and India (Uk Aid, 2010; Hay et al., 2010).

 

• World Malaria Report

Malaria is a preventable and treatable mosquito-borne disease, whose main victims are children under five years of age in Africa. The World Malaria Report 2012 summarizes data received from 104 malaria-endemic countries and territories for 2011. Ninety-nine of these countries had on-going malaria transmission.

According to the World Malaria Report, 2011, there were 655,000 malaria deaths worldwide in 2010, compared to 781,000 in 2009 (WHO, 2000; World Malaria Report, 2011). It has been estimated that 91% of deaths in 2010 were in the African Region, followed by the South-East Asia (6%) and Eastern Mediterranean Regions (3%) (World Malaria Report, 2011). About 86% of deaths globally were in children under 5 years of age (World Malaria Report, 2011). Of the 35 countries that accounted globally for approximately 98% of malaria deaths, 30 were located in sub-Saharan Africa, with four countries (Nigeria, Democratic Republic of the Congo, Uganda and Ethiopia) alone accounting for approximately 50% of deaths on the continent as shown in Fig. 1 (World Malaria Report, 2011). However, a recent systematic analysis by Murray et al has estimated that the global malaria deaths increased from 995,000 in 1980 to a peak of 1,817,000 in 2004, decreasing to 1,238,000 (929,000 — 1,685,000) in 2010 (almost double of the WHO estimate for the same year) (Murray et al., 2012). This study estimated more deaths in individuals aged 5 years or older than has been estimated in previous studies: 435,000 (307,000 — 658,000) deaths in Africa and 89,000 (33,000 — 177,000) deaths outside of Africa in 2010 (Murray et al., 2012).

 

Fig 1: World malaria burden (World Malaria Report, 2011)

 

 

 

1.1.2 Malaria in Children

Children under five years of age are the most vulnerable group affected by malaria. There were an estimated 660,000 malaria deaths around the world in 2010, of which approximately 86% were in children under five years of age (WHO, 2011).

In high transmission areas, partial immunity to the disease is acquired during childhood. In such settings, the majority of malarial disease, and particularly severe disease with rapid progression to death, occurs in young children without acquired immunity. Severe anaemia, hypoglycaemia and cerebral malaria are features of severe malaria more commonly seen in children than in adults (WHO, 2012).

 

1.1.3 Malaria Parasite Life Cycle

       Malaria parasite has a complex, multistage life cycle occurring within two living beings, the vector mosquitoes and the vertebrate hosts. The survival and development of the parasite within the invertebrate and vertebrate hosts, in intracellular and extracellular environments, is made possible by a toolkit of more than 5,000 genes and their specialized proteins that help the parasite to invade and grow within multiple cell types and to evade host immune responses (Laurence et al., 2002; Brian et al., 2008). The parasite passes through several stages of development such as the sporozoites (Gr. Sporos = seeds; the infectious form injected by the mosquito), merozoites (Gr. Meros = piece; the stage invading the erythrocytes), trophozoites (Gr. Trophes = nourishment; the form multiplying in erythrocytes), and gametocytes (sexual stages) and all these stages have their own unique shapes and structures and protein complements. The surface proteins and metabolic pathways keep changing during these different stages that help the parasite to evade the immune clearance, while also creating problems for the development of drugs and vaccines (Laurence et al., 2002).

 

1.1.3.1 Sporogony within the Mosquitoes

Mosquitoes are the definitive hosts for the malaria parasites, wherein the sexual phase of the parasite's life cycle occurs. The sexual phase is called sporogony and results in the development of innumerable infecting forms of the parasite within the mosquito that induce disease in the human host following their injection with the mosquito bite. When the female Anopheles draws a blood meal from an individual infected with malaria, the male and female gametocytes of the parasite find their way into the gut of the mosquito. The molecular and cellular changes in the gametocytes help the parasite to quickly adjust to the insect host from the warm-blooded human host and then to initiate the sporogonic cycle. The male and female gametes fuse in the mosquito gut to form zygotes, which subsequently develop into actively moving ookinetes that burrow into the mosquito midgut wall to develop into oocysts. Growth and division of each oocyst produces thousands of active haploid forms called sporozoites. After the sporogonic phase of 8–15 days, the oocyst bursts and releases sporozoites into the body cavity of the mosquito, from where they travel to and invade the mosquito salivary glands. When the mosquito thus loaded with sporozoites takes another blood meal, the sporozoites get injected from its salivary glands into the human bloodstream, causing malaria infection in the human host. It has been found that the infected mosquito and the parasite mutually benefit each other and thereby promote transmission of the infection. The Plasmodium-infected mosquitoes have a better survival and show an increased rate of blood-feeding, particularly from an infected host (Heather and Andrew, 2004; Carolina et al., 2005; Hill, 2006).

 

• Schizogony in the Human Host

Man is the intermediate host for malaria, wherein the asexual phase of the life cycle occurs. The sporozoites inoculated by the infested mosquito initiate this phase of the cycle from the liver, and the latter part continues within the red blood cells, which results in the various clinical manifestations of the disease (Ashley et al., 2008).

 

• Pre-erythrocytic Phase – Schizogony in the Liver

The sporozoites that find a blood vessel reach the liver within a few hours. It has recently been shown that the sporozoites travel by a continuous sequence of stick-and-slip motility, using the thrombospondin-related anonymous protein (TRAP) family and an actin–myosin motor (Jake et al., 2006; Lucy et al., 2007; Sylvia et al., 2009). The sporozoites then negotiate through the liver sinusoids, and migrate into a few hepatocytes, and then multiply and grow within parasitophorous vacuoles. Each sporozoite develop into a schizont containing 10,000 –30,000 merozoites (or more in case of P. falciparum) (Amino et al., 2006; Malcolm and Micheal, 2006; Kebaier et al., 2009). The growth and development of the parasite in the liver cells is facilitated by a a favorable environment created by the The circumsporozoite protein of the parasite (Miguel et al., 2006; Agam et al., 2007). The entire pre-eryhrocytic phase lasts about 5–16 days depending on the parasite species: on an average 5-6 days for P. falciparum, 8 days for P. vivax, 9 days for P. ovale, 13 days for P. malariae and 8-9 days for P. knowlesi. The pre-erythrocytic phase remains a “silent” phase, with little pathology and no symptoms, as only a few hepatocytes are affected (Ashley et al., 2008). This phase is also a single cycle, unlike the next, erythrocytic stage, which occurs repeatedly. The merozoites that develop within the hepatocyte are contained inside host cell-derived vesicles called merosomes that exit the liver intact, thereby protecting the merozoites from phagocytosis by Kupffer cells. These merozoites are eventually released into the blood stream at the lung capillaries and initiate the blood stage of infection thereon (Olivier et al., 2008).

 

• Erythrocytic Schizogony – Centre Stage in Red Cells

Red blood cells are the ‘centre stage' for the asexual development of the malaria parasite. Within the red cells, repeated cycles of parasitic development occur with precise periodicity, and at the end of each cycle, hundreds of fresh daughter parasites are released that invade more number of red cells. The merozoites released from the liver recognize, attach, and enter the red blood cells (RBCs) by multiple receptor–ligand interactions in as little as 60 seconds. This quick disappearance from the circulation into the red cells minimises the exposure of the antigens on the surface of the parasite, thereby protecting these parasite forms from the host immune response (Alan and Brendan, 2006; Brian et al., 2008; Olivier et al., 2008). The invasion of the merozoites into the red cells is facilitated by molecular interactions between distinct ligands on the merozoite and host receptors on the erythrocyte membrane. P. vivax invades only Duffy blood group-positive red cells, using the Duffy-binding protein and the reticulocyte homology protein, found mostly on the reticulocytes. the more virulent P. falciparum uses several different receptor families and alternate invasion pathways that are highly redundant. Varieties of     Duffy binding-like (DBL) homologous proteins and the reticulocyte binding-likehomologous proteins of P. falciparum recognize different RBC receptors other than the Duffy blood group or the reticulocyte receptors. Such redundancy is helped by the fact that P. falciparum has four Duffy binding-like erythrocyte-binding protein genes, in comparison to only one gene in the DBL-EBP family as in the case of P. vivax, allowing P. falciparum to invade any red cell (David et al., 2002; Ghislaine et al., 2009).

The process of attachment, invasion, and establishment of the merozoite into the red cell is made possible by the specialized apical secretory organelles of the merozoite, called the micronemes, rhoptries, and dense granules. The initial interaction between the parasite and the red cell stimulates a rapid “wave” of deformation across the red cell membrane, leading to the formation of a stable parasite–host cell junction. Following this, the parasite pushes its way through the erythrocyte bilayer with the help of the actin–myosin motor, proteins of the thrombospondin-related anonymous protein family (TRAP) and aldolase, and creates a parasitophorous vacuole to seal itself from the host-cell cytoplasm, thus creating a hospitable environment for its development within the red cell. At this stage, the parasite appears as an intracellular “ring” (Alan and Brendan, 2006; Jurgen et al., 2007).

Within the red cells, the parasite numbers expand rapidly with a sustained cycling of the parasite population. Even though the red cells provide some immunological advantage to the growing parasite, the lack of standard biosynthetic pathways and intracellular organelles in the red cells tend to create obstacles for the fast-growing intracellular parasites. These impediments are  overcome by the growing ring stages by several mechanisms: by restriction of the nutrient to the abundant hemoglobin, by dramatic expansion of the surface area through the formation of a tubovesicular network, and by export of a range of remodeling and virulence factors into the red cell (Olivier et al., 2008). Hemoglobin from the red cell, the principal nutrient for the growing parasite, is ingested into a food vacuole and degraded. The amino acids thus made available are utilized for protein biosynthesis and the remaining toxic heme is detoxified by heme polymerase and sequestrated as hemozoin (malaria pigment). The parasite depends on anaerobic glycolysis for energy, utilizing enzymes such as pLDH, plasmodium aldolase etc. As the parasite grows and multiplies within the red cell, the membrane permeability and cytosolic composition of the host cell is modified (Kiaran, 2001; Virgilio et al., 2003). These new permeation pathways induced by the parasite in the host cell membrane help not only in the uptake of solutes from the extracellular medium but also in the disposal of metabolic wastes, and in the origin and maintenance of electrochemical ion gradients. At the same time, the premature hemolysis of the highly permeabilized infected red cell is prevented by the excessive ingestion, digestion, and detoxification of the host cell hemoglobin and its discharge out of the infected RBCs through the new permeation pathways, thereby preserving the osmotic stability of the infected red cells (Kiaran, 2001; Virgilio et al., 2003).

The erythrocytic cycle occurs every 24 hours in case of P. knowlesi, 48 h in cases of P. falciparum, P. vivax and P. ovale and 72 h in case of P. malariae. During each cycle, each merozoite grows and divides within the vacuole into 8–32 (average 10) fresh merozoites, through the stages of ring, trophozoite, and schizont. At the end of the cycle, the infected red cells rupture, releasing the new merozoites that in turn infect more RBCs. With sunbridled growth, the parasite numbers can rise rapidly to levels as high as 10¹³ per host (Brian et al., 2008).

A small proportion of asexual parasites do not undergo schizogony but differentiate into the sexual stage gametocytes. These male or female gametocytes are extracellular and nonpathogenic and help in transmission of the infection to others through the female anopheline mosquitoes, wherein they continue the sexual phase of the parasite's life cycle. Gametocytes of P. vivax develop soon after the release of merozoites from the liver, whereas in case of P. falciparum, the gametocytes develop much later with peak densities of the sexual stages typically occurring 1 week after peak asexual stage densities (Louis et al., 2002; Sasithon et al., 2008).

 

1.1.4 Pathogenic Basis of Malaria

Millions of children die from malaria in Africa every year (Snow et al., 1999). But the clinical outcome of an infection in a child depends on many factors (parasite, host, geographical and social factors). These factors, often ill defined, determine the outcome in each child (Marsh et al., 1995; English, 1996). The top priority must be disease prevention because of the inability of the mothers to access or afford optimal treatment, and the ever evolving drug resistance. Prevention may be effected through vector control such as insecticide-treated bed nets or through the development of antimalarial vaccines (Taylor et al., 1993).

Over the past 10 years, there have been several key shifts in our understanding of what constitutes severe malaria, and these shifts define the issues in pathogenesis that need to be explored to develop better treatments for sick children (English, 1997). The first shift is the increasing recognition that severe malaria is a disorder that affects several tissues and organs, even when the most marked manifestations may seem to involve a single organ such as the brain. In particular, metabolic acidosis, often profound, has been recognized as a principal pathophysiological feature that cuts across the classical clinical syndromes of cerebral malaria and severe malarial anaemia (Marsh et al., 1995). It is the single most important determinant of survival and leads directly to a common, but previously poorly recognized, syndrome of respiratory distress (Taylor et al., 1993). In most cases, this is predominantly (but not exclusively) a lactic acidosis (English, 1996). There are several causes of lactic acidosis in children with severe malaria, from increased production of lactic acid by parasites (through direct stimulation by cytokines) to deceased clearance by the liver; however, most important by far is probably the combined effects of several factors that reduce oxygen delivery to tissues (English, 1997).

A key feature of the biology of Plasmodium falciparum is its ability to cause infected red blood cells (RBCs) to adhere to the linings of small blood vessels. Such sequestered parasites cause considerable obstruction to tissue perfusion. In addition, in severe malaria there may be marked reductions in the deformability of uninfected RBCs (Miller et al., 1971; Dondorp et al., 2000). The pathogenesis of this abnormality is not clear, but its strong correlation with acidosis suggests that it may be involved in compromising blood flow through tissues. Individuals affected with malaria are often dehydrated and relatively hypovolaemic, which potentially exacerbates microvascular obstruction by reducing perfusion pressure. The destruction of RBCs is also an inevitable part of malaria, and anaemia further compromises oxygen delivery (English, 1996).

The second and related shift in our concept of severe malaria is the realization that there is no simple one-to-one correlation between the clinical syndromes and the pathogenic processes. Thus, severe anaemia may arise from many poorly understood mechanisms including acute haemolysis of uninfected RBCs and dyserythropoiesis, as well as through the interaction of malarial infection with other parasite infections and with nutritional deficiencies (Newton, 1997). For many desperately sick children a simple ‘one pathogen/one disease’ model is not adequate, as bacteraemia caused by common pathogens may be present with acute malaria and may be a factor in mortality (Prada et al., 1993; Berkley et al., 1999). Even the rigorously defined syndrome of cerebral malaria is used to describe children who have arrived at the point of coma through different routes. In many of these children, coma seems to be a response to overwhelming metabolic stress rather than a primary problem in the brain. Such children are often profoundly acidotic and may regain consciousness remarkably quickly after appropriate resuscitation (English et al., 1996).

Similarly, it has been recognized that a significant proportion of children in coma are, in fact, experiencing covert status epilepticus (Crawley, 1996), which responds rapidly to appropriate anticonvulsant therapy. The pathogenesis of this condition is unknown, but again the speed of resolution argues against classical views of pathogenesis. The picture that emerges is one in which many processes lead to a common outcome. These distinctions are much more than academic: they have direct implications for therapy, and they also identify the research issues needed to improve therapy for sick children (English et al., 1996; Berkley et al., 1999).

 

1.1.5 Pathophysiology of Severe Malaria in Children

In Africa, malaria continues to be one of the most important causes of childhood morbidity and mortality. Most deaths, in children admitted to hospital with severe malaria, occur within the first 24 h (Newton and Krishna, 1998). In other words the majority of children die of the complications of severe malaria before they can benefit treatment. Therefore, improvements in outcome will require the implementation of supportive therapies directed at treating complications and correcting disordered physiology.

However, it has now been recognised that this model is too simplistic and in recent years severe malaria has been regarded as a complex syndrome affecting many organs. It has also become apparent that metabolic acidosis, often manifesting as respiratory distress, is an important component of the severe malaria syndrome (Taylor et al., 1993; Krishna et al., 1994; Allen et al., 1996; Marsh et al., 1996). Furthermore, metabolic acidosis has been demonstrated to be the best independent predictor of a fatal outcome, in both adults and children (Waller et al., 1995; Allen et al., 1996; Marsh et al., 1996). This has led to a change in the understanding of the processes underlying severe malaria and an appreciation that severe malaria comprises systemic functional derangements resulting from the host–parasite interaction (Marsh, 1999).

 

1.1.6 Cytokine-associated Neutrophil Extracellular Traps and Antinuclear Antibodies in Plasmodium falciparum

Pathogenesis in humans infected with Plasmodium falciparum involves a complex multifactorial immune system response to the parasite as well as to host cell and tissue damage. Although much is known about the immunological response to falciparum infection (Malaguarnera and Musumeci, 2002; Stevenson and Riley, 2004; Good et al., 2005), relationships between immunocompetence (Millington et al., 2006) and disease severity remain poorly understood. Patient age (Nussenblatt et al., 2001), genetics (Griffiths et al., 2005), vitamin sufficiency (Chillemi et al., 2004; Nzila et al., 2005; Gregson and Plowe, 2005), gravidae (Many et al., 2001; Moore et al., 2004; Bulgan et al., 2005), control of oxidative stress (Pabon et al., 2003; Llurba et al., 2004; Muller, 2004), and factors related to the availability of complement proteins and their receptors (Taylor et al., 2000; Stoute et al., 2003; Ciurana et al., 2004; Li et al., 2004; Kravitz et al., 2005; Roumenina et al., 2005; Dernellis and Panaretou, 2006) all affect immunocompetence, as does the presence of immunosuppressive (Millington et al., 2006)  and autoimmune factors (Daniel-Ribeiro, 2000).

The levels of certain cytokines associated with falciparum malaria can provide clues to the immune system reaction, but analyses of cytokine levels alone can yield paradoxical results concerning the protection and pathology of the underlying highly integrated responses (Kern et al., 1989; Means and Krantz, 1992; Jennings et al., 1997; Kurtzhals et al., 1998; Dalton et al., 2000; May et al., 2000; Nussenblatt et al., 2001; Griffiths et al., 2001; Malaguarnera et al., 2002; Stoute et al., 2003; Muller, 2004; Chaiyaroj et al., 2004; Coltel et al., 2004; Wassmer et al., 2006). An IFN-γ-Th1-dependent immune response in the mouse model, for example, has been associated with both immunoprotection (Stevenson and Riley, 2004) and immunopathology (Rae et al., 2004). Likewise, elevated CRP levels can both activate the classical complement cascade and yet provide protection for endothelial cells from membrane attack complex deposition through up regulation of surface receptor expression to counter the effects of the activated cascade (Li et al., 2004).

The immune response to falciparum infection may depend not only on the cytokine profile but also on hematologic activity. Recently, a novel activity of neutrophils, formation of neutrophil extracellular traps (NETs), has been described (Brinkmann et al., 2004; Gupta et al., 2005; Urban et al., 2006; Festjens et al., 2006; Fuchs et al., 2007; Golstein and Kroemer, 2007). NETs can bind and kill a variety of microbes (Brinkmann et al., 2004; Urban et al., 2006), but NET formation has not been described previously as a response to falciparum malaria infection.

               

1.2 Biochemistry of Plasmodium falciparum

The malaria parasite, like all organisms, must acquire nutrients from the environment and convert these nutrients to other molecules or energy (i.e., catabolism). These other molecules and the energy are then used to maintain the homeostasis of the parasite, and in the growth and reproduction of the parasite (i.e., anabolism). Both anabolic and catabolic processes are catalyzed by enzymes. Growing and reproducing organisms require high levels of macromolecules and other biochemicals for the maintenance of cellular structure and function. The malaria parasite needs to acquire these biochemicals and precursors from the host (Banerjee et al., 2002).

The unique life cycle and resulting microenvironments of the parasite has led to the evolution of metabolic pathways which differ from the human host (Curley et al., 1994; Dalal and Klemba, 2007). It may be possible to exploit these unique pathways and enzymes in the design of therapeutic strategies. For example, many anti-malarials are known to affect the food vacuole which is a special organelle for the digestion of host of host hemoblobin (Eggleson et al., 1999; Egan, 2008).

 

 

• Hemoglobin Degradation and the Food Vacuole:

The malaria parasite requires amino acids for the synthesis of its proteins. The three sources of amino acids are: de novo synthesis, import from host plasma, and digestion of host haemoglobin (Florent et al., 1998). Haemoglobin is an extremely abundant protein in the erythrocyte cytoplasm and serves as the major source of amino acids for the parasite (Ginsburg et al., 1999).

 

• Ingestion of Host Cytoplasm:

During the early ring stage, the parasite takes up the host cell stroma by pinocytosis as shown in Fig. 2 below (note ppm = parasite plasma membrane) resulting in double membrane vesicles. The inner membrane, which corresponds to the parasitophorous vacuole membrane (PVM) surrounding Plasmodium, rapidly disappears and the digestion of hemoglobin takes place within these small vesicles during the early trophozoite stage (Goldberg et al., 1991; Goldberg, 2005). As the parasite matures, it develops a special organelle, called the cytostome, for the uptake of host cytoplasm and the small pigment-containing vesicles fuse to form a large food vacuole. (Gametocytes do not form the large food vacuole and are characterized by small pigment-containing vesicles found throughout their cytoplasm.) Double-membrane vesicles pinch off from the base of the cytostome and fuse with the food vacuole. The inner membrane (originally the PVM) is lysed and the hemoglobin is released into the food vacuole (Hempelmann, 2007).

 

 

Fig. 2: Ingestion of host cytoplasm

 

• Proteases and the Food Vacuole:

The food vacuole is an acidic compartment (pH 5.0-5.4) that contains protease activities. In this regard the food vacuole resembles a lysosome, except other acid hydrolases (eg., nucleases) have not been identified. Presumably other acid hydrolases are not needed since the microenvironment of the erythrocyte is almost exclusively protein, and in particular, hemoglobin (Klemba et al., 2004).

 

Several distinct protease activities, representing three of the four major classes of proteases (plasmepsins, falcipains and falcilysin) have been identified in the food vacuole. The digestion of hemoglobin probably occurs by a semi-ordered process involving the sequential action of different proteases (Goldberg, 2005). Several plasmespsin genes have been identified in the genome of P. falciparum and four of these appear to function in the food vacuole (Banerjee et al., 2002). Plasmepsin-1 and plasmepsin-2 are the best characterized and both are capable of cleaving undenatured hemoglobin between phenylalanine and leucine residues located at positions 33 and 34 on the alpha-globin chains. These residues are located in a conserved domain known as the hinge region, which is believed to be crucial in stabilizing the overall structure of hemoglobin. Cleavage at this site presumably causes the globin subunits to dissociate and partially unfold. This unfolding will expose additional protease sites within the globin polypeptide chains. The other plasmepsins, as well plasmepsin-1 and plasmepsin-2, and the falcipains are then able to further degrade these large globin fragments. It has been suggested that falcipain-2 (Shenai et al., 2000), and possibly falcipain-3 (Sijwali et al., 2001), are capable of digesting either native hemoglobin and therefore may also participate in the initial cleavage of hemoglobin.

 

• Food Vacuole Proteases:

Initially no food vacuole associated exopeptidase activity could be identified within the food vacuole (Kolakovich et al., 1997). However, recently two amino peptidases have been found in the food vacuole (Dalal and Klemba, 2007) which can convert the peptides into amino acids. In addition, a dipeptidyl aminopeptidase (DPAP) activity has been identified within the food vacuole (Klemba et al., 2004). It is postulated that the DPAP may remove dipeptides from the N-termini of the peptides generated through the actions of the various endopeptidases in the food vacuole and then the amino peptidases can convert these to amino acids.

 

A neutral amino peptidase activity has been identified in cytoplasm of several Plasmodium species (Curley et al., 1994; Florent et al., 1998). This implies that the digestion of the small peptides also takes place in the parasite cytoplasm, and therefore must be pumped out of the food vacuole. Pfmdr-1 has been localized to the food vacuole membrane and is a member of the ATP-binding cassette (ABC) transporter superfamily. Some ABC transporters function to translocate polypeptides across membranes. For example, the STE6 gene of yeast transports the a-type mating factor (a 12 amino acid peptide). Pfmdr-1 can complement the STE6 gene (Volkman et al., 1995) indicating that it could function to pump small peptides into the parasite cytoplasm.

 

 

 

 

1.2.1 Detoxification of Heme and Reactive Oxygen Intermediates

Digestion of hemoglobin also releases heme. Free heme is toxic due to its ability to destabilize and lyse membranes, as well as inhibiting the activity of several enzymes. Three, and possibly four, mechanisms by which heme is detoxified have been identified:

• sequestration of the free heme into hemozoin, or the malarial pigment;
• a degradation facilitated by hydrogen peroxide within the food vacuole;
• a glutathione-dependent degradation which occurs in the parasite's cytoplasm;
• and possibly a heme oxygenase which has been found in P. berghei (rodent parasite) and P. knowlesi (simian parasite), but not P. falciparum (Rosenthal et al., 1988).

Both the hemozoin formation pathway and the degradative pathways probably function simultaneously with 25-50% of the free heme being converted into hemozoin and the remainder being degraded (Ginsburg et al., 1999). However, some studies suggest that up to 95% of the free iron released during hemoglobin digestion is found in hemozoin (Egan, 2008). X-ray crystallography and spectroscopic analysis indicates that hemozoin has the same structure as b-hematin (Pagola et al., 2000). b-hematin is a heme dimer formed via reciprocal covalent bonds between carboxylic acid groups on the protoporphyrin-IX ring and the iron atoms of two heme molecules. These dimers interact through hydrogen bonds to form crystals of hemozoin. Therefore, pigment formation is best described as a biocrystallization, or biomineralization, process (Hempelmann, 2007; Egan, 2008). The mechanism of hemazoin formation is not known, but recently a protein that may catalyze the formation of hemozoin has been described (Klemba et al., 2004). Lipids may also participate in the process in that lipid bodies have been observed within the food vacuole and hemozoin is associated with lipids (Egan, 2008).

 

A portion of the free heme may be degraded into non-toxic metabolites. Three potential processes have been described: in the food vacuole a hydrogen peroxide mediated oxidation of the porphyrin ring leads to its opening and subsequent breakdown; some of the heme translocates across the food vacuole membrane into the host cytoplasm where it is oxidized by reduced glutathione (GSH); and a heme oxygenase activity has been identified in some non-human malaria parasites. However, the role these processes play in the degradation of heme is not known (Florent et al., 1998).

Chloroquine and other 4-aminoquinolines inhibit pigment formation, as well as the heme degradative processes (Ginsburg et al., 1999), and thereby prevent the detoxification of heme. The free heme destabilizes the food vacuolar membrane and other membranes and leads to the death of the parasite. The fact that the biocrystallization of heme is a unique process to the parasite and not found in the host accounts for the high therapeutic index of such drugs in the absence of drug resistance. Many other anti-malarials target the food vacuole indicating the importance of this organelle and its various functions to the survival of the parasite (Florent et al., 1998).

The iron bound to hemoglobin is primarily in the ferrous state (Fe²⁺). Release of the heme results in iron being oxidized to the ferric state (Fe³⁺). Electrons liberated by this oxidation of iron promote the formation of reactive oxygen intermediates (ROI) such as superoxide anion radicals and hydrogen peroxide. ROI can cause cellular damage. Superoxide dismutase (SOD) and catalase are cellular enzymes that function to prevent oxidative stress by detoxifying the superoxide and hydrogen peroxide, respectively. Both of these activities are found in the food vacuole and may have been obtained from the host during ingestion of the erythrocyte cytoplasm. Hydrogen peroxide can also be exported into the parasite cytoplasm where it is detoxified by catalase and glutathione peroxidase. Some of the hydrogen peroxide produced as a result of the Fe²⁺-Fe³⁺conversion may also used for the peroxidative degradation of heme (Kolakovich et al., 1997; Dalal and Klemba, 2007).

 

1.2.2 Biochemistry and Molecular Biology of Malaria Parasite: Pyrimidine Biosynthetic Pathway

Four malarial species infect humans, the most deadly being Plasmodium falciparum. In the fight against this disease, there is an urgent need to develop new antimalarials and an effective vaccine because of widespread resistance to current chemotherapeutic agents (Nchinda, 1998; Ridley, 2002). At present, the complete nucleotide sequences of the 23-megabase nuclear genome of P. falciparum consists of 14 chromosomes, encoding about 5,300 genes, and is the most (A+T)-rich genome sequenced to date (Gardner et al, 2002). In the post-genomic era, metabolism of the malaria parasite has been mapped based on the current knowledge of parasite biochemistry and on pathways known to occur in other eukaryotes (Gardner et al, 2002). Some metabolic pathways in the parasite are unique and found to be markedly different from the mammalian host, e.g., hemoglobin catabolism, fatty acid synthesis, folate biosynthesis and metabolism of nucleic acids (Ridley, 2002). Understanding of metabolic functions should illuminate new chemotherapeutic targets for drug development, including the identification of target(s) for drugs in current use. Recently, it has been proposed that the pyrimidine metabolic pathway may be a target for the design of new antimalarial drugs (Krungkrai et al, 1992; Krungkrai, 1993; McRobert and McConkey, 2002; Ridley, 2002).

The erythrocytic malarial parasites require purines and pyrimidines for DNA/RNA synthesis and other metabolic pathways during exponential multiplication in the human host. They use preformed purines from the host and must synthesize pyrimidines de novo (Gero and O’Sullivan, 1990). The parasites lack thymidine kinase, which is responsible for salvaging the preformed thymidine from the host (Reyes et al, 1982). Several lines of evidence suggest that there are some key differences between malarial parasites and the human host in the pyrimidine pathway. The first six enzymes of the pathway, catalyzing the conversion of HCO₃ -, ATP, L-glutamine and Laspartate to uridine 5´ monophosphate (UMP), are demonstrated in both P. falciparum and a rodent parasite P. berghei (Reyes et al, 1982; Rathod and Reyes, 1983; Gero and O’Sullivan, 1990; Krungkrai et al, 1990; 1991; 1992; Krungkrai, 1995). Some genes encoding the six enzymes are partially sequenced, in order, from the first to the sixth step; these are CPSII (carbamyl phosphate synthase II, CPSII) (Flores et al, 1997), ATC (aspartate transcarbamylase, ATC), DHO (dihydroorotase, DHO), DHOD (dihydroorotate dehydrogenase, DHOD) (LeBlanc and Wilson, 1993), OPRT (orotate phosphoribosyltransferase, OPRT), and OMPDC (orotidine 5´-monophosphate decarboxylase, OMPDC) (van Lin et al, 2001). The human host has five enzymes out of the six, associated into two different multifunctional proteins, in that a single gene CPSII ATC-DHO encoded the first three enzymes and another gene OPRT-OMPDC encoded the last two enzymes (Jones, 1980).

• Complication of Plasmodium falciparum Malaria

Severe falciparum malaria is defined by the demonstration of asexual forms of P. falciparum in a patient with a potentially fatal manifestation or complication of malaria in which other diagnoses have been excluded (Andrej et al., 2003). Even though, the complications are almost unique to P. falciparum, infection that does not mean that all cases of P. falciparum malaria invariably develop complications. The case fatality of P. falciparum malaria is around 1 per cent and this accounts for 1 to 3 million deaths per year all over the world. 80% of these

deaths are caused by cerebral malaria (Njuguna and Newton, 2004).

In 1990, the World Health Organization (WHO) established criteria for severe malaria and these were revised in the year 2000 to include other clinical manifestations and laboratory values that portend a poor prognosis based on clinical experience in semi-immune patients as shown in Table 1 below. The major complications of severe malaria include cerebral malaria, pulmonary edema, acute renal failure, severe anemia, and/or bleeding. Acidosis and hypoglycemia are the most common metabolic complications. Any of these complications can develop rapidly and progress to death within hours or days (Andrej et al., 2003).

The presentation of severe malaria varies with age and geographical distribution. In areas of high malaria transmission, severe malaria mainly affects children under five years of age. The mortality rate is higher in adults than in children but African children develop neuro-cognitive sequelae following severe malaria more frequently. In children, the complications include metabolic acidosis (often caused by hypovolaemia), hypoglycaemia, hyperlacticacidaemia, severe anaemia, seizures and raised intracranial pressure and concomitant bacterial infections occur more frequently. In adults, renal failure and pulmonary oedema are more common causes of death (Njuguna and Newton, 2004).

 

1.2.4 Prevalence and Management of Plasmodium falciparum Malaria among Infants and Children

Malaria is the most prevalent tropical disease in the world today. Each year, it causes disease in approximately 650 million people and kills between one and three million, most of them, young children in Sub-Saharan Africa (Hay et al., 2004). Nigeria is known for high prevalence of malaria and it is a leading cause of morbidity and mortality in the country. Available records show that at least 50 per cent of the population of Nigeria suffers from at least one episode of malaria each year and this accounts for over 45 per cent of all out- patient visits (Ojurongbe et al., 2007). Malaria infection during the first five years of life is a major public health problem in tropical and subtropical regions throughout the world (Trampuz et al., 2003; Greenwood et al., 2005).

In endemic areas, treatment is often less satisfactory and the overall fatality rate for all cases of malaria can be as high as one in ten (Mockenhaupt et al., 2004). For reasons that are poorly understood, but which may be related to high intracranial pressure, children with malaria frequently exhibit abnormal posturing, a sign indicating severe brain damage (Idro et al., 2007; Mockenhaupt et al., 2004). Malaria has been found to cause cognitive impairments, especially in children. Malaria causes widespread anaemia during a period of rapid brain development and also direct brain damage and this neurologic damage results from cerebral malaria to which children are more vulnerable (Boivin, 2002). Over the longer term, developmental impairments have been documented in children who have suffered episodes of severe malaria (Trampuz et al., 2003).

 

1.3 Hypoglycaemia in Childhood Malaria

Hypoglycaemia is a medical emergency that involves an abnormally diminished concentration of glucose in the blood (Ferry and Allen, 2010). It can produce a variety of symptoms and effects but the principal problems arise from an inadequate supply of glucose to the brain, resulting in impairment of function (neuroglycopenia) (Osier et al., 2003; Planche et al., 2005. Effects can range from mild dysphoria to more serious issues such as seizures, unconsciousness, and (rarely) permanent brain damage or death (Graz et al., 2008).

In the paediatric age group, and particularly among under-fives, hypoglycaemia is a common metabolic problem encountered in association with a variety of diseases (Solomon et al., 1994; Elusiyan et al., 2006; Zijlmans et al., 2009). In countries with limited resources, undernutrition (Wharton, 1991), infectious diseases (Bondi, 1991), delayed presentation in hospital (Hendrickse, 1991), administration of potentially toxic herbal concoctions (Bondi, 1991; Hendrickse, 1991; Solomon et al., 1994), and lack of facilities for diagnosis may increase the frequency of occurrence of hypoglycaemia.

Hypoglycaemia is a well recognized complication of Plasmodium falciparum malaria with or without treatment with quinine and it is associated with increased mortality and neurologic sequelae, particularly among under-fives (Planche et al., 2005; Krause, 2007; Kapse, 2009). In these patients, it is difficult to identify hypoglycaemia from clinical examination alone, because all the signs of hypoglycaemia may be mimicked by those of malaria (Osier et al., 2003; Kapse, 2009; Ferry and Allen, 2010). In addition, hypoglycaemia is one of the markers of disease severity in children with falciparum malaria (Hendrickse, 1991; Osier et al., 2003; Planche et al., 2005). In the light of the above, hypoglycaemia should always be considered, assessed and, if present, treated in severe malaria. Given that hypoglycaemia is amenable to inexpensive and readily available treatment, various clinicians have recommended that children with falciparum malaria be monitored frequently for hypoglycaemia (Osier et al., 2003; Planche et al., 2005). However, regular monitoring has been ignored by clinicians (Osier et al., 2003) despite the fact that hypoglycaemia is associated with serious neurological sequelae when detection is delayed or treatment inadequate (Ferry and Allen, 2010).

Various pathogenetic mechanisms have been postulated to explain the occurrence of hypoglycaemia in children with falciparum malaria. In acute falciparum malaria, there is increased glucose turnover due to increased glucose consumption both by the host and the parasite (Davies et al., 1993; WHO, 2000), with the host’s requirement being considerably greater (WHO, 2000). Fasting reduces glycogen stores rapidly even in well nourished children, the presence of high substrate levels (lactate and alanine) and absence of ketosis in many children with hypoglycaemia suggest that other factors than starvation might be involved (WHO, 2000).

 

1.3.1 Sublingual Sugar for Hypoglycaemia in Children with Severe Malaria

Hypoglycemia is a common determining factor of poor prognosis for children with severe malaria in sub-Saharan Africa. Intravenous dextrose administration is rarely available. Oral mucosal delivery may be an alternative to parenteral administration. A randomized, clinical trial was performed in Burkina Faso among moderately hypoglycemic children, comparing sublingual sugar administration with oral water, oral sugar, and dextrose infusion administrations (Barennes et al., 2005; Graz et al., 2008).

The sublingual administration of sugar proved to be effective among moderately hypoglycemic children. It is a simple and promising method to control hypoglycemia among children in both developing and developed countries. This pediatric practice should be investigated in more detail among children with severe malaria (Barennes et al., 2005).

 

1.4          Lactic Acidosis in Childhood Malaria

Lactic acidosis is a physiological condition characterized by low pH in body tissues and blood (acidosis) accompanied by the buildup of lactate, especially D-lactate, and is considered a distinct form of metabolic acidosis (Luft, 2001). The condition typically occurs when cells receive too little oxygen (hypoxia), for example, during vigorous exercise. In this situation, impaired cellular respiration leads to lower pH levels. Simultaneously, cells are forced to metabolize glucose anaerobically, which leads to lactate formation. Therefore, elevated lactate is indicative of tissue hypoxia, hypoperfusion, and possible damage. Lactic acidosis is characterized by lactate levels >5 mmol/l and serum pH <7.35 (Robergs et al., 2004).

Children who die of malaria present with overlapping syndromes that are associated with impaired consciousness and metabolic complications (Planche and Krishna, 2005). These metabolic complications include hypoglycaemia, hyperlactataemia, and metabolic acidosis. If hypovolemia significantly contributes to the acidosis seen in severe malaria (Maitland et al., 2003; Maitland and Newton, 2005), then aggressive fluid resuscitation may be useful in the early treatment of severe malaria. However, because of dangers inherent in aggressive fluid management, such as the complications of pulmonary oedema, hypokalaemia (Maitland et al., 2003), and brain swelling (Newton et al., 1997), the role of fluid therapy in malaria remains controversial (Planche and Krishna, 2005; Planche, 2005) and urgently needs investigation.

 

1.4.1      Lactate Levels in Severe Malarial Anaemia

In severe malaria, metabolic acidosis is one of the most important determinants of survival (Krishna et al., 1994). In African children with malaria, the clinical syndrome of respiratory distress usually reflects an underlying metabolic acidosis associated with lactic acidaemia (English et al., 1997). This syndrome is an important independent, clinical prognostic marker for poor outcome (Marsh et al., 1995).

The pathophysiology of metabolic acidosis is complex. The direct contribution of P. falciparum to the final lactate concentration, through anaerobic glycolysis in the parasite itself, is likely to be small (Vander et al., 1990). More significantly, an inadequate supply of oxygen to tissues may follow from severe anaemia and provoke a metabolic shift within host cells to anaerobic glucose metabolism and increased lactic acid production. In addition, the flow of blood through the microcirculation may be impeded by adherence of infected erythrocytes to the endothelium of post-capillary venules and/or increased rigidity of uninfected cells (Dondorp et al., 1997). Lactate may not in itself be sufficient to cause acidaemia but the inhibition of oxidative metabolism in the context of an ongoing inflammatory response will cause protons (H⁺) to accumulate and eventually lead to metabolic acidosis (English et al., 1997). These pathophysiological pathways suggest that the syndrome of lactic acidosis may be associated with the total parasite burden during acute infection.

Classically, parasitaemia has been associated with the severity of clinical disease (Field and Hodgkin, 1937). However, the relationship is weak and the association of parasite density with specific syndromes of severe disease is less clear. Haemozoin (Hz) or malaria pigment, the final product of digested host haemoglobin, is often seen in circulating leucocytes and may be a surrogate marker for acute or chronic parasite load (Day et al., 1996).

However, the clinical significance of Hz has only been investigated quite recently. Nguyen and colleagues found an association between Hz-containing neutrophils (HCN) and outcome and between HCN and Hz-containing monocytes (HCM) and hyperparasitaemia, shock and hypoglycaemia (Nguyen et al., 1995). In African children with severe malaria, Hz containing leucocytes were associated with severe malaria (Metzger et al., 1995; Lyke et al., 2003), cerebral malaria (Amodu et al., 1998) and anaemia (Luty et al., 2000; Lyke et al., 2003). More recently, Casals-Pascual and colleagues have reported the association of HCM, free Hz and bone marrow Hz with severe malarial anaemia (Casals-Pascual et al., 2006).

Severe disease has also been associated with high levels of pro-inflammatory cytokines. Raised levels of TNF-α and IFN-γ are more frequently observed in children suffering from severe malarial disease than those suffering from mild disease or those with asymptomatic infections (Malaguarnera and Musumeci, 2002). On the other hand, high levels of IL-10 have been associated with protection from anaemia (Kurtzhals et al., 1998). Finally, low levels of IL-12 have been found in children with severe, compared to mild disease (Luty et al., 2000; Malaguarnera et al., 2002) and IL-12 may promote a Th1 type response and have other regulatory functions in modulation of an inflammatory response. The immune response to parasites may contribute not only to parasite clearance and amelioration of disease but also to immunopathology and physiological disturbance.

In addition, the relationship between the parasites, cytokines and outcome of infection may depend on the direct effect(s) of Hz on leucocytes. Hz-containing macrophages from the peripheral circulation have increased secretion of inflammatory cytokines (Pichyangkul et al., 1994) or anti-inflammatory cytokines (Keller et al., 2004). Moreover, the number of Hz-containing monocytes is associated with serum TNF-α level in children with malaria (Luty et al., 2000).

 

1.5          Transport of Lactate and Pyruvate in Plasmodium falciparum Malaria

The intraerythrocytic form of the human malaria parasite, Plasmodium falciparum, lacks a functional citric acid cycle and is largely reliant on glycolysis to fulfil its very substantial energy requirements (Sherman, 1998). Human erythrocytes infected with mature (trophozoite-stage) parasites consume glucose up to two orders of magnitude faster than normal, uninfected erythrocytes (Scheibel et al., 1979; Roth et al., 1982), ultimately converting it to lactic acid (Pfaller et al., 1982; Vander Jagt et al., 1990). Accumulation of lactic acid within the parasite cytosol would lead to a chronic decrease in intracellular pH (pH_i) and would threaten the osmotic stability of the cell. It would also interfere with the oxidation of glycolytically derived NADH to NAD⁺, which occurs through the conversion of pyruvate to lactate via lactate dehydrogenase (Sherman, 1998).

For all of these reasons it is important that the parasite have an efficient means of clearing lactic acid from its cytosol. In many cells, monocarboxylates such as lactate are transported across the plasma membrane via H⁺-linked monocarboxylate transporters (MCTs), which operate with a stoichiometry of 1:1 (i.e. 1 H⁺ per monocarboxylate anion; reviewed in (Halestrap and Price, 1999). A family of mammalian MCTs has been identified, as have related proteins from non-mammalian species. MCTs are susceptible to inhibition by a range of compounds, including the bioflavonoid phloretin, substituted aromatic monocarboxylates such as cinnamic acid derivatives, the thiol reagent p chloromercuribenzenesulphonate (pCMBS) and anion transport inhibitors such as niflumic acid (Sherman, 1998).

 

1.6          Anaemia in Childhood Malaria

Anaemia is a decrease in number of red blood cells (RBCs) or less than the normal quantity of hemoglobin in the blood (Benoist et al., 2008). In heavily endemic malaria areas, it is almost inevitable that malarial infection will be associated with anaemia, although malaria may not be the prime cause of it (Facer, 1994; Murphy and Oldfield, 1996). Anaemia an indicator of both poor nutrition and poor health is a common and sometimes serious complication of P. falciparum infection (Nussenblatt and Semba, 2002; Benoist et al., 2008). Anaemia impairs normal development in children and it constitutes a major public health problem in young children in the developing world with wide social and economic implication (Le Cornet et al., 1998). The highest prevalence of anaemia exists in the developing world where its causes are multi factorial (Tolentino and Friedman, 2007). The complex aetiology of anaemia involves the interaction between multiple factors including nutritional deficiencies, genetic red blood cell disorders, infectious diseases particularly malaria, hookworm and human immunodeficiency virus infections (van Eijk et al., 2002).

 

1.6.1      Severity of Anaemia in Children Diagnosed with Plasmodium falciparum           Malaria

Malaria-induced anaemia (MIA), characterised by low haemoglobin levels, is  one of the life-threatening complications of childhood malaria (Newton et al.,1997; Asobayire et al., 2001). It is a serious public health problem in the tropics. In addition to the many deaths caused by severe cases, a strong association has been established between anaemia and impaired development of physical, motor, cognitive, immunological and neurological functions in children, especially when it occurs in the very early years of life (Walter et al., 1989; Eden, 2003). The possible long-term implications of childhood anaemia, which are thought to include permanent or irreversible psychomotor and mental retardation (Lozoff et al., 2000), add to the health consequences of MIA in the long term.

 

1.7          Typhoid and Malaria Co-Infection

Malaria and typhoid fever are among the most endemic diseases in the tropics. Both diseases have been associated with poverty and underdevelopment with significant morbidity and mortality. An association between malaria and typhoid fever was first described in the medical literature in the middle of the 19th century, and was named typhomalarial fever by the United States Army (Smith, 1992). However, by the end of 19^th century, laboratory tests had eliminated this theory as they found that it was either one thing or the other, or in rare instances, co-infection with both Salmonella typhi and the Plasmodium species (Smith, 1992). In the last two decades, this relationship between the two diseases has been substantiated by studies from Africa and India (Samal and Sahu, 1991; Ammah et al., 1999; Ohanu et al., 2003; Kanjilal et al., 2006; Sur et al., 2006).

Typhoid fever is a prolonged, severe, systemic, life threatening illness caused by the bacterium Salmonella enterica, sub-species enterica serotype typhi commonly called Salmonella typhi (Wain et al., 1998). Cases are more likely to be seen in areas like India, South and Central America and Africa with rapid population growth (Willke et al., 2002). Typhoid and paratyphoid fevers are usually transmitted via the faecal -oral route, either directly from person to person or by ingestion of food or water contaminated with faeces or urine (Brooks et al., 2001; Chart, 2004). Previous reports have shown the presence of significant salmonella antibodies in persons living in Nigeria (Agbonlahor et al., 1992; Mohammed et al., 1992; Isibor and Onwuzuruigbo, 1999).

 

1.8          Aim and Objectives of the Study

 

1.8.1      Aim of the Study

This study was aimed at determining the prevalence and severity of hypoglycaemia and lactic acidosis in Nigerian children diagnosed with Plasmodium falciparum malaria.

 

1.8.2      Specific Objectives of the Study

The aim of this study was to be achieved through the following specific objectives;

1. To determine the prevalence of Plasmodium falciparum malaria in out-patients at specialist children hospital in Nsukka.
2. To determine the prevalence and severity of hypoglycaemia in the subjects diagnosed with Plasmodium falciparum
3. To determine the prevalence and severity of lactic acidosis in the subjects.
4. To determine the prevalence of anaemia in the subjects.
5. To determine the relationship between the parasite load and some biochemical parameters (blood glucose, blood lactate and haemoglobin concentration).
6. To determine the relationship between blood glucose, lactate and haemoglobin concentrations in childhood malaria.

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