Anemia is a condition that occurs when the amount of haemoglobin in a person’s drops below normal .and a decrease in haemoglobin is often associated with a decrease in the number of red blood cells and haematocrit which may be cause by malaria while Malaria is a major public health problem in Nigeria where it accounts for more cases and deaths than any other country in the world. Malaria is a risk for 97% of Nigeria’s population 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. (WHO, 2011). Malaria is a disease cause by a plasmodium parasite transmitted by the bite of infected mosquitoes. One of the key contributory factors to the development and progression of its complication is that malaria may develop anaemia deficiency and malaria can generate free radicals that can possibly promote the development of anaemia and oxidative stress; a condition characterized by increase production of free radicals or impaired antioxidant defense system. An antioxidant which help in the removal of free radicals thereby preventing oxidative stress. Hence, this study was aimed to investigate the relationship between malaria infection, anemia deficiency and antioxidant status of patients visiting ESUTH Enugu. The study was conducted at ESUTH Enugu, were the formal concept patients was obtained from patients. A total 101 patients were recruited for the study, a questionnaire was use to collected a basic characteristic of the patients after which a blood sample was collected to measure the haemoglobin conc, haematocrit conc, vitamin C, catalase, SOD( superioxide dimutase) of a patients were screened through various tests conducted in laboratory. According to the results obtained, 58 (57.4%) patients were anaemic while (43(42.6%) were non anaemic. The haemoglobin (10.33±0.194) / (30.38±0.57), haematocrit (13.66±0.24)/ (40.07±0.73) levels were significant lower in anaemic patients than in the non anaemia patients (p- <0.001). While only the catalase showed a significant difference which elucidated that the catalase was higher in anaemic patents (1.45±0.03) compare to the non anaemia patients (1.35±0.03). Vitamin C and SOD conc level showed no significant between anaemic and non anaemic patients. There was no relationship between anaemia and malaria (p = 0.827). Also, there was no interaction between anaemia and malaria to influence the antioxidant indices as there was no significant difference among the anaemic and non-anaemic patients (p > 0.05). Findings from this study showed that there was no relationship between anaemia, malaria and antioxidant status of patient visiting ESUTH, Enugu.

TABLE OF CONTENT

Title page - - - - - - - - - - - i

Certification - - - - - - - - - - - ii

Dedication - - - - - - - - - - - iii

Acknowledgement - - - - - - - - - - iv

Table of content- - - - - - - - - - vi

List of tables- - - - - - - - - - - x

List of figures - - - - - - - - - - xi

Abstract - - - - - - - - - - - xii

CHAPTER 1

Introduction Abstract - - - - - - - - - 1

1.1 Background of study - - - - - - - - - 1

1.2 Statement of the problem - - - - - - - - 4

1.3 Hypothesis - - - - - - - - - 3

1.4 Aim of the study - - - - - - - - - 5

1.5 Objectives of the study - - - - - - - - 5

1.6 Limitation of the study - - - - - - - - 5

CHAPTER TWO

2.0 Literature Review - - - - - - - - 6

2.2 Global view of malaria - - - - - - - - 7

2.3 Introduction to malaria - - - - - - - - 8

2.4 Cause of Anemia - - - - - - - - - 8

2.5 Etiology of Malaria - - - - - - - - 9

2.6 The life circle of malaria Parasite - - - - - - - 10

2.6.1 Phases of Malaria - - - - - - - - - 11

2.6.2 The Pre-erythrocytic Stage - - - - - - - - 11

2.6.3 Erythrocytic Stage - - - - - - - - - 12

2.6.4 Sexual Spoorogony Phase - - - - - - - - 13

2.7 Transmission of malaria parasite - - - - - - - 13

2.8 Complication of Malaria/Anemia, Oxidative stress and antioxidant - - 13

2.9 Factors that Determine the Occurance of Malaria - - - - - 15

2.9.1 Climate - - - - - - - - - - - 15

2.10 Sings and Symptoms of Malaria - - - - - - - 16

2.11 Malaria diagnosis - - - - - - - - - 17

2.12 Rapid Diagnostic Test (RDT) - - - - - - - - 17

2.12.1 Microscopic examination - - - - - - - - 17

2.13 Malaria, Diseases and Oxidative Stress - - - - - - 18

2.14 Oxidative stress in Plasmodiun Falciparum infected erytrosyts - - - 18

2.15 Oxidative changing Plasmodiun - - - - - - - 20

2.16 Oxidative changes in the host induced by Plasmodiun - - - - 20

2.17 Meganism of oxidative stressing in human host - - - - - 21

2.18 Treatment of malaria/ Anaemia and management of oxidative stress - - 23

2.18.1 Deficiency - - - - - - - - - - 25

2.18.2 Metabolism - - - - - - - - - - 25

2.18.3 Food source - - - - - - - - - - 25

2.18.4 Infants requirements - - - - - - - - - 26

2.18. 5 Children and adolescence - - - - - - - - 26

2.18.6 Adult - - - - - - - - - - - 27

2.18.7 Pregnancy and Lactation - - - - - - - - 29

2.19 The role of Ascorbic acid in biological pathways- - - - - 29

CHAPTER THREE

3.0 Materials and Methods - - - - - - - - - 31

3.1 Consumables - - - - - - - - - - 31

3.2 Equipments - - - - - - - - - - 31

3.3 Reagents - - - - - - - - - - - 31

3.4 Study population and design - - - - - - - - 32

3.5 Study duration - - - - - - - - - - 32

3.6 Inclusion and exclusion criteria - - - - - - - - 32

3.7 Data collection - - - - - - - - - - 32

3.8 Blood collection - - - - - - - - - - 32

3.9 Sample collection - - - - - - - - - 32

3.10 Malaria diagnosis by rapid diagnostic test (RDT) - - - - - 33

3.10.1 Drawing blood with a tool - - - - - - - - 33

3.10.2 How to read and interpret RDT Test Result - - - - - - 34

3.11 Microscopic test for malaria - - - - - - - - 34

3.14 Diagnosis of Haemoglobin and Haematocrt - - - - - - 35

3.15 Determination of Vitamin C - - - - - - - - 35

3.16 Determination of Catalase activity - - - - - - - 36

3.17 Determination of superoxide Dismutase (SOD) activity - - - - 36

3.18 Statistical analysis - - - - - - - - - 36

CHAPTER FOUR

Result - - - - - - - - - - - 38

CHAPTER FIVE

5.0 Discussion - - - - - - - - - - 43

5.1 Conclusion - - - - - - - - - - 44

Reference - - - - - - - - - - 45

CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the Study

Malaria is a common and life threatening disease in many tropical and subtropical areas and the term malaria originates from medieval Italian malaran: mala-aria “bad air”; the disease was formerly called ague or marsh fever due to its association with swamps and marshland. The term first appeared in the English literature about 1829. (Breeveld et al., 2012) There are currently over 100 countries and territories where there is high risk of malaria transmission.

Malaria is one of the most important tropical infectious diseases. The annual worldwide incidence is estimated to be 300–500 each year with a mortality of between one and three million people. (Asante et al., 2004).

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), and Malaria is the 3rd leading cause of death for children under five years worldwide.

Malaria in Nigeria Malaria is a major public health problem in Nigeria where it accounts for more cases and deaths than any other country in the world. Malaria is at risk to 97% of Nigeria’s population. The remaining 3% of the population live in the malaria free highlands. (Kremsner et al., 200). There are an estimated 100 million malaria cases with over 300,000 deaths per year in Nigeria. This compares with 215,000 deaths per year in Nigeria from HIV/AIDS. Malaria contributes to an estimated 11% of maternal mortality. (WHO, 2012).

Malaria accounts for 60% of outpatient visits and 30% of hospitalizations among children under five years of age in Nigeria. Malaria has the greatest prevalence of 27.6 percent, in children age 6 to 59 months in the South East region. (Abubakar et al., 2002).

Malaria is a significant global problem. In 2015, there were 214 million cases of the disease worldwide, killing about 438,000 people. In other areas of the world, malaria causes substantial morbidity, especially in the rural areas of some countries in Asia and South America , and these countries are visited by more than 125 million international travelers every year and malaria is most often cause by travel to and from endemic areas. (Abubakar et al., 2002).

Malaria is a mosquito-borne infectious disease affecting humans and other animals caused by parasitic protozoan’s (a group of single-celled microorganisms) belonging to the Plasmodium types like Plasmodium, falciparum, Plasmodium, vivax, Plasmodium, ovale, Plasmodium, malariae and transmitted to humans/ animals by the bite of infected mosquitoes (WHO, 2014). The malaria parasite, entering the blood after an infective mosquito bite, which will infects/invades the red bloods cells. At the end of that infection cycle, red blood cell ruptures. The process lowers the amount of red blood cells and can cause anaemia which is low haemoglobin levels, frequently leading to anaemia. Plasmodium facliparum causes the most severe and profound anemia Malaria infection in human by plasmodium species is associated with a reduction with a significant risk of death, this cannot be explained simply by the direct destruction of parasitized red blood cells.

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. (Becker K et al., 2004). It was observed that erythrocytes infected with Plasmodium. falciparum produced OH• radicals and H₂O₂ about twice as much compared to normal erythrocytes. Higher level of these free radicals can lead to oxidative stress. (Becker K et al., 2004).

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 as well as by malaria parasite. The consequence of oxidative stress once it is high, it can attack cellular membrane lipids, causing damage to cells and tissues such as the brain, metabolic disorders or inherited disease affecting electron transport chain.

Oxidative stress during malaria is considered useful to the patient in the fight against the intra-erythrocytic parasite (Gilbert D.L. 1981). Studies have been described in which induction of oxidative stress by treatment with pro-oxidants proved to be effective against the infection. On the other hand ROS play a role in the pathology of malaria (Jayshree et al., 1993). Excessive oxidative stress particularly at unprescribed sited (e.g. vascular lining, blood brain barrier) can damage the defense system. This is however, controlled by intra- and extracellular anti-oxidants systems, which may fail during disease. Treatment with anti-oxidants reinforces these systems and protects the patient, especially during the life threatening phase of the disease (Rice-Evans et al., 1992).

In malaria infections, the most probable target of free radical generated by malaria parasite is the red blood cell since it is where the parasite resides. Thus, though malaria parasite infection also damage the red blood cell during its asexual stage multiplication, free radicals produced by the parasite may also contribute. Thus, it may be suggested that anaeamia which is defined by the reduction of haemoglobin in blood below normal may result from the parasite damaging the red blood cell as well as free radical generation Anemia is a condition that occurs when the amount of haemoglobin in a person’s drops below normal .and a decrease in haemoglobin is often associated with a decrease in the number of red blood cells and haematocrit. Haemoglobin is contained with RBCs and it is necessary to transport and delivery of oxygen from the lungs to the rest of the body. Without a sufficient supply of oxygen, many tissues and organs throughout the body can be adversely affected. People with anemia may experience faigue weakness / tiredness, and may lack energy, breathlessness. groups , However, certain people have increase risk of developing anemia Anaemia fairly common condition affecting both men and women of all ages, race and ethnic. These include people with diets poor in irons and vitamins, chronic diseases such as kidney disease and inflammatory bowel disease. Therefore, increased level of anaemia in malaria may be due to the influence of free radical induced oxidative stress.

Antioxidants are molecules that inhibit or quench free radical reactions and delay or inhibit cellular damage (Adam-Vizi, V. (2005). 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 (Adam-Vizi, V. (2005). The major enzymatic antioxidants are superoxide dismutase, catalyze and glutathione peroxidase. Superoxide dismutase exists as a copper, zinc-enzyme (SOD1) that is found in the cytoplasm or a manganese containing enzyme that is located in mitochondria (SOD2). These enzymes catalyze the one-electron dismutation of superoxide (O₂·) to hydrogen peroxide (2O₂ + 2H⁺ → H₂O₂ + O₂). Catalyse is an iron-dependent enzyme that directly decomposes hydrogen peroxide to water (2H₂O₂ → 2H₂O + O₂). Furthermore, glutathione peroxidases (GPXs) are a family of enzymes that reduce a variety of organic and inorganic hydroperoxides to the corresponding hydroxyl derivatives in the presence of glutathione (GSH). In this process, GSH is converted to an oxidized disulfide (2GSH + H₂O₂ → GS-SG + 2H₂O).

The other way of categorizing the antioxidants is based on their solubility in the water or lipids. The antioxidants can be categorized as water-soluble antioxidants. The water-soluble antioxidants (e.g. vitamin C) are present in the cellular fluids such as cytosol, or cytoplasmic matrix.

1.2 Statement of the problem

Malaria remains one of the leading causes of morbidity and mortality worldwide and in sub- Saharan Africa. (WHO, 2012). Mortality from malaria is due to complication arising as a result of severe infections usually caused by Plasmoduim Falciparum. Studies on mortality have shown that deaths occur predominantly among young children/ some adult and mortality rates among patients with an illness severe enough to warrant hospitalization are consistently high with case fatality rates varying from 5% to 30% in Nigeria, malaria is hyper endemic and presents a serious health problem in the country. It is also a leading cause of deaths in the country and accounts for over 40% of out-patient attendance with annual reported cases and children less than five years are the most affected. A study conducted by Ministry of Health in 2006 showed that more than 17 million of Nigerian’s over twenty million people are infected with malaria/anaemia every year, with cost of $95 million for treatment. Despite the importance of Plasmoduim Falciparum. As a human pathogen, the patho-phyrsiologic basis of its infection is not well understood. Parasitic infections such as malaria in host organisms often lead to anaemia and oxidative stress condition which is a disturbance in the balance between the production of ROS and antioxidant defenses .generation of free radicals as a result of oxidative stress and other reactive species in vivo leads to extensive damage in parasite bio- molecules such as DNA, lipids and proteins. It has also been shown that the parasites are vulnerable to oxidative stress during their erythrocytic life stages. In erythrocytes, Plasmoduim Falciparum. Encounters enhanced oxidative stress, resulting largely from its digestion of haemoglobin and thus, its redox balance becomes fragile. Superoxide (O₂-) is normally produced when oxidized haemoglobin is exposed to the acid environment of the food vacuole, and can therefore be considered as the major source of ROS. Inside the parasite, regardless of its origin, O₂- is dismutated by superoxide dismutase (SOD) to H₂O₂ (Makani, J. et al., 2010). Even though studies have been carried out on Plasmoduim Falciparum and ROS, the focus has mainly been on the pathological effect of these radicals.

1.3 Hypothesis

Malaria may promote oxidative stress and anaemia and reduce antioxidants.

1.4 Aim of the study

The study was undertaken to evaluate the relationship between anaemia, malaria infection, and antioxidant status of patients visiting Enugu State University Teaching Hospital (ESUTH) in Enugu Nigeria.

1.5 Objective of the study

To determine the presence of malaria parasite infection in patients
Determine haemoglobin concentration and haemoglobin to assess anaemia in infected patients.
Determine antioxidant indices (SOD, and Vitamin C) among malaria patients.