Malaria is a significant public health problem in the world especially in in sub-Saharan Africa. One of the key contributory factors to the development and progression of malaria and its complications is oxidative stress, a condition characterized by increased production of free radicals or impaired antioxidant defence system. Glucose-6-phosphate dehydrogenase (G-6-PD) produces NADPH which intends regenerate reduced glutathione (GSH), an antioxidant which helps in the removal of free radicals thereby preventing oxidative stress, Hence, this study was aimed to investigate the relationship between G-6-PD, oxidative stress and malaria infection inpatients visiting ESUT Teaching Hospital Parklane, Enugu. The recruited patientsfollowing their informed consent were screened for malaria by RDT and Microscopy methods and their baseline parameters including age, gender, etc. were obtained using a questionnaire. Whole blood was collected and used for the determination of malaria infection, oxidative stress, lipid peroxidation and protein oxidation, anaemia and as well as the G-6-PD status in patients. A total of 101 patients were recruited including 30 male and 71 female among which 86 had malaria positive while 15 tested malaria negative. Comparing RDT and Microscopy techniques in diagnosing Malaria, showed RDT to have a low performance in in diagnosing malaria using microscopy as standardwith a sensitivity of 10.47% and of accuracy 23.76%. All the baseline characteristics of study participants was not significantly different (p = 0.946) among the malaria and non- malaria patients. Among the G6PD deficient patients,17.9%were found to be anaemicwhile 13.1% were non-anaemic whereas among the non-deficient patients, 39.3% were anaemic while 29.8% were non-anaemic. As such, there was no significant relationship (p = 0.946) betweenG6PD deficiency and among the malaria patients.Comparison of anaemia and oxidative stress indices among malaria patients showed significantly (p<0.05) low level of haemoglobin and haematocrit concentrations, but there no significant difference (p>0.05) of MDA and protein oxidation level between anemic and non-anaemic patients with malaria. Interaction between anaemia and G6PD deficiency on study parameters, showed no significant (p<0.05) relationship on haemoglobin, haematocrit, MDA and protein oxidation level in malaria patients. In conclusion, this study showed the no association between anaemia, oxidative stress and G-6-PD deficiencyamong malaria patients. Further studies are needed to ascertain these findings as oxidative stress is implicated in the pathogenesis of malaria.

TABLE OF CONTENTS

Title page i

Certification ii

Dedication iii

Acknowledgment iv

Abstract v

Table of contents vi

List of Figure ix

List of table x

Chapter 1: Introduction

1. Background of study 1
2. Statement of the problem 6
3. Hypothesis 6
4. Aim 6
5. Research objectives 7

Chapter 2: Literature review

2.1 Malaria 8

2.1.1 History of Malaria 8

2.1.2 Biochemical background of malaria 9

2.1.3 Types of malaria diagnosis 11

2.1.6 Management of malaria 18

2.2 Glucose -6-Phosphate Dehydrogenase 20

2.2.1 Enzymology of glucose -6-Phosphate Dehydrogenase 22

2.2.2 Glucose -6- Phosphate Dehydrogenase Deficiency 23

2.3 Oxidative Stress 24

2.3.1 ROS and RNS production 26

2.3.2 Diabetes (Type1 and 2) and oxidative Stress 31

2.3.3 Role of Oxidative Stress 33

Chapter 3: Materials and methods 36

3.1 chemicals 36

3.1.1 Reagents 36

3.2 Preparation of reagent 37

3.3 Study population and parameters 37

3.4 Ethical considerations 37

3.5 Methods 38

3.5.1 Test of fasting blood sugar 38

3.5.2 Determination of Catalase activity 38

3.5.3 Estimation of lipid peroxidation 39

3.5.4 Super Oxide Dismutase (SOD) 39

3.5.5 Glucose-6-phosphate dehydrogenase deficiency assay 40

3.6 Statistical analysis 40

Chapter 4:Results and Discussion

4.1 Result 42

4.2 Discussion 49

Chapter 5: Conclusion /Recommendation

5.1 Conclusion 51

5.2 Recommendation 51

Reference 52

Appendix 68

CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of study

Malaria is a mosquito-borne infectious disease affecting humans and other animals caused by parasitic protozoans (a group of single-celled microorganisms) belonging to the Plasmodium type (WHO, 2014). According to the World Health Organization (WHO), malaria is a significant public health problem in more than 100 countries and causes an estimated 200 million infections each year, with more than 500 thousand deaths annually. Over 90% of these deaths occur in sub-Saharan Africa, where the disease is estimated to kill one child every 30 seconds (WHO, 2011). In other areas of the world, malaria causes substantial morbidity, especially in the rural areas of some countries in Asia and South America. Malaria causes symptoms that typically include fever, tiredness, vomiting, and headaches. In severe cases it can cause yellow skin, seizures, coma, or death (Caraballo, 2014). Symptoms usually begin ten to fifteen days after being bitten, If not properly treated, people may have recurrences of the disease months later. The disease is most commonly transmitted by an infected female Anopheles mosquito. The mosquito bite introduces the parasites from the mosquito's saliva into a person's blood (WHO, 2014). The parasites travel to the liver where they mature and reproduce. Five species of Plasmodium can infect and be spread by humans. (Caraballo, 2014). Most deaths are caused by Plasmodium falciparum.

The role of oxidative stress during malaria infection is still unclear. Some authors suggest a protective role, whereas others claim a relation to the physiopathology of the disease (Sohail et al., 2007). However, recent studies suggest that the generation of reactive oxygen and nitrogen species (ROS and RNS) associated with oxidative stress, plays a crucial role in the development of systemic complications caused by malaria. Malaria infection induces the generation of hydroxyl radicals (OH^•) in the liver, which most probably is the main reason for the induction of oxidative stress and apoptosis (Guha et al., 2006). Additionally, Atamna et al. (1993) observed that erythrocytes infected with P. falciparum produced OH^• radicals and H₂O₂ about twice as much compared to normal erythrocytes. Higher level of this free radicals can lead to oxidative stress.

Oxidative stress, termed as an imbalance between production and elimination of reactive oxygen species (ROS) leading to plural oxidative modifications of basic and regulatory processes, can be caused in different ways. Increased steady-state ROS levels can be promoted by drug metabolism, over-expression of ROS-producing enzymes, or ionizing radiation, as well as due to deficiency of antioxidant enzymes. The consequence of oxidative stress once it is high, it can cause damage to the brain, metabolic disorders affecting electron transport chain. Reactive oxygen species (ROS), generated by endogenous and exogenous sources, cause significant damage to macromolecules, including DNA (Salmon et al., 2004).

Furthermore, Spermatozoa are highly vulnerable to oxidative attack because they lack significant antioxidant protection due to the limited volume and restricted distribution of cytoplasmic space in which to house an appropriate armoury of defensive enzymes. In particular, sperm membrane lipids are susceptible to oxidative stress because they abound in significant amounts of polyunsaturated fatty acids. Susceptibility to oxidative attack is further exacerbated by the fact that these cells actively generate reactive oxygen species (ROS) in order to drive the increase in tyrosine phosphorylation associated with sperm capacitation. However, this positive role for ROS is reversed when spermatozoa are stressed. Under these conditions, they default to an intrinsic apoptotic pathway characterised by mitochondrial ROS generation, loss of mitochondrial membrane potential, caspase activation, phosphatidylserine exposure and oxidative DNA damage. In responding to oxidative stress, spermatozoa only possess the first enzyme in the base excision repair pathway, 8-oxoguanine DNA glycosylase. This enzyme catalyses the formation of abasic sites, thereby destabilising the DNA backbone and generating strand breaks. Because oxidative damage to sperm DNA is associated with both miscarriage and developmental abnormalities in the offspring, strategies for the amelioration of such stress, including the development of effective antioxidant formulations, are becoming increasingly urgent (Aitken et at., 2016).

The process of lipid peroxidation involves a complex chain reaction utilizing the interaction of oxygen-derived species with polyunsaturated fatty acids (e.g. docosahexaenoic acid, linoleic acid and arachidonic acid), resulting in highly reactive electrophilic aldehydes and free radicals (Esterbauer et al., 1991). This process is extremely detrimental to cellular functions as it disrupts membrane integrity, fluidity and function (Esterbauer et al., 1991). Lipid peroxidation is a self-propagating process involving initiation and propagation steps which continue through an ongoing free radical chain reaction until termination occurs. The retina is particularly prone to lipid peroxidation since it is highly enriched in polyunsaturated fatty acids (PUFAs) (Catalase). The predominant PUFA in photoreceptor outer segments is docosahexanoic acid which is the most unsaturated fatty acid in the body. Lifelong accumulation of chronic oxidative damage will lead to dysfunction in retinal cells and increase their susceptibility to exogenous and endogenous insults eventually culminating in loss of visual function and cell death (Esterbauer et al.,1991). Malaria infection has been found to be associated with lipid peroxidation accompanying reduction in antioxidant capacity of the infected patients especially Plasmodium falciparum infection. Instantaneous reduction in antioxidant potency in tandem with increased lipid peroxidation is also observed to be equally accountable for development of oxidative stress in malaria patients (Das and Nanda, 1999; Upadhyay et al., 2011; Egwunyenga et al., 2004). Any infection, including malaria, activates the immune system of body thereby causing release of reactive oxygen species as an antimicrobial action (Kulkarni et al., 2003). In addition to host’s immune system, malaria parasite also stimulates certain cells in production of reactive oxygen species thereby resulting in hemoglobin degradation (Loria et al., 1999; Pradines et al., 2005). One of the major reasons for development of malarial anemia seems to be oxidative stress (Das and Nanda, 1999; Kremsner et al., 2000) while changes in micronutrient metabolism alter disease progression and severity (Singotamu et al., 2006).

Proteins are the largest constituent of the cellular milieu and are frequent targets of oxidative damage (Stadtman, 2004). Protein oxidation can involve direct reaction with amino acids, cleavage of the polypeptide chain, and conversion of the protein to derivatives that are highly sensitive to proteolytic degradation. It has also been established that all of these protein modifications can be mediated by metal-catalyzed oxidation systems. All amino acid residues of proteins are potential targets for oxidation by HO· or by H₂O₂ in the presence of metal ions. For example, oxidation of tyrosine residues is damaging to the red blood cells, as this amino acid is converted to a 3,4-dihydroxyphenylanine derivative, which itself can undergo redox cycling to generate further ROS (Sugiura and Ichinose, 2011).

Antioxidants are molecules that inhibit or quench free radical reactions and delay or inhibit cellular damage (Young et al., 2001). Though the antioxidant defenses are different from species to species, the presence of the antioxidant defense is universal. Antioxidants exists both in enzymatic and non-enzymatic forms in the intracellular and extracellular environment. . Enzymatic antioxidants work by breaking down and removing free radicals. The antioxidant enzymes convert dangerous oxidative products to hydrogen peroxide (H₂O₂) and then to water, in a multi-step process in presence of cofactors such as copper, zinc, manganese, and iron. Non-enzymatic antioxidants work by interrupting free radical chain reactions (Young et al.,2001).

The antioxidants can also be categorized according to their size, the small-molecule antioxidants and large-molecule antioxidants. The small-molecule antioxidants neutralize the ROS in a process called radical scavenging and carry them away.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathological disease in humans. This disease is described as a widespread, heritable, X-chromosome linked abnormality (Reclose et al., 2000). It is estimated that it affects approximately 400 million people worldwide (Daloii et al., 2004). This disease is seen most frequently in approximately all of Africa, Asia, and the countries near the mediterranean Sea (Frank, 2005). G6PD enzyme was demonstrated to play an active role in survival of erythrocytes. It is known that in the pentose phosphate pathway of erythrocytes, glucose-6 phosphate dehydrogenase (G6PD) enzyme provides the production of NADPH and Glutathione (GSH). GSH, produced by pentose phosphate pathway can react with H₂O₂ and reduce it to H₂O. This prevents the generation of oxidative stress within red blood cells; oxidative stress can be induced in erythrocytes whose G6PD enzymes are deficient. In this situation, GSH is not produced and H₂O₂ is not reduced to H₂O, leading to oxidative stress and hemolysis.

1.2 Justification / Need for study

The need for this study is to carefully ascertain whether G6pD deficiency has an impact on oxidative stress and malaria infection. To known if G6pD can promote malaria infection in patient. This study is equally designed to give information about the prevalence of G6PD and malaria infection among malaria patient in Enugu metropolis.

1.3 Statement of the problem

So many reasons have been attributed to susceptibility of G-6-PD deficient patients to malaria infection. Recall that the pentose phosphate pathway is essential in producing enough NADPH capable of reducing oxidized glutathione but when there is a default in the production of enough NADPH from PPP as the case may be in G-6-PD deficient patients, leading to a decrease in the level of reduced glutathione which leads to an increased in oxidative stress – a possible risk factor in the development of malaria infection will be complicated.

1.4 Aim of the study

This study was undertaken to evaluate the relationship between oxidative stress among malaria patients visiting Enugu State University Teaching Hospital, Parklane, Enugu Nigeria.

1.5 Objective of the study

1. To screen patients for malaria infection.

2. To assess anaemia in malaria patients.

3. To assess G6PD deficiency in malaria patients.

4. To assess oxidative stress indices (lipid peroxidation and protein oxidation) in malaria patients.