ABSTRACT

The increased clinical state of malaria infection may be due to poor nutritional status most especially as a result of micronutrients deficiency. Micronutrients play vital role both in combating anaemia and other adverse effects of malaria infection in humans and animals in developing resistance against the disease. This research was aimed at establishing the effect of malaria parasite on neutrophil-lymphocyte ratio, platelet-lymphocyte ratio and some essential plasma electrolytes (calcium, magnesium and iron) as well as vitamins (vitamin B₁₂ and folate) of children. Ethical approval was gotten from ministry of Health, Benin City, Edo State. Test group was recruited from the paediatric ward of Central Hospital, Benin City. A questionnaire, written in English was administered to the parents of the subjects to fill. An informed consent was given to the parents or guardian of the children before sample was collected Blood sample was collected from two hundred (n = 200) children within the ages of l – 10 years as test group who are positive for malaria parasites while the control group were one hundred (100) children above l0yrs of age who do not have malaria parasites. Thick blood film was used to ascertain the level of parasitaemia using giemsa stain, vitamin B₁₂ was done using electroluminescence technique, folic acid was analysis was done using ELISA technique, while iron, magnesium and calcium was spectrophotometrically analyzed. Results gotten in this research shows that PCV decreased significantly compared to the control group, (p<0.05). White blood cell increased (p<0.05) compared to the control, Plasma calcium, magnesium and iron decreased significantly; there was decrease in folate and vitamin B₁₂. Micronutrients are not only necessary in the regeneration of heamolyzed red cells during malaria infection, but also served as antioxidants hence protecting the red cells against damage by malaria toxins. It is therefore of tremendous importance to assess micronutrients status of children with malaria.

TABLE OF CONTENTS

TITLE PAGE. i

CERTIFICATION.. ii

DEDICATION.. iii

ACKNOWLEDGEMENT. iv

TABLE OF CONTENTS. v

LIST OF TABLES. x

LIST OF FIGURES. xi

ABSTRACT. xii

CHAPTER ONE. 1

INTRODUCTION.. 1

1.1    Background of Study. 1

1.2    Justification of Study. 4

1.3    Aim of Study. 4

1.4    Specific Objective. 5

1.5    Research Design. 5

1.6    Ethical consideration. 5

1.7    Research Hypothesis 5

1.8    Questionnaire/informed consent 6

1.9    Sample size. 6

CHAPTER TWO.. 7

2.0    LITERATURE REVIEW.. 7

2.1    Malaria. 7

2.2    Epidemiology. 7

2.3    Malaria parasites 10

2.3.1  Life cycle of Malaria Parasite. 11

2.3.2  Signs and Symptoms of Malaria in Children. 13

2.3.3  Consequences of Malaria in Children. 13

2.3.4  Severe Malaria Anaemia in Children. 14

2.3.5  Laboratory Diagnosis of Malaria. 14

2.4    Micronutrients 15

2.4.1  Functions of Micronutrients 17

2.4.2  Micronutrients Deficiency in children. 18

2.4.3  The Prevalence of Micronutrient Deficiency in Diet Plans 19

2.5    Vitamin B₁₂ (Cobalamin) 20

2.5.1  Structure of Vitamin B₁₂ 20

2.5.2  Metabolism of Vitamin B₁₂; Absorption. 21

2.5.3  Mechanism of Absorption of Vitamin B₁₂ 22

2.5.4  Transportation, Storage and Excretion of Vitamin B₁₂ 23

2.5.5  Functions of Vitamin B₁₂ 25

2.5.6  Deficiency of Vitamin B₁₂ 25

2.5.7  Laboratory Assessment status of Vitamin B₁₂ 26

2.6    Folic Acid (Folate) 26

2.6.1  Structure of Folic acid. 26

2.6.2  Formation of Folic acid (F.H₄) 27

2.6.3  Biosynthesis of Folic acid. 29

2.6.4  Functions of Folic acid. 30

2.6.5  Deficiency of Folic acid. 30

2.6.6  Laboratory Assessment Status of Folic acid. 31

2.7    Iron (Fe) 31

2.7.1  Essential Iron. 32

2.7.2  Storage Iron. 32

2.7.3  Absorption of Iron and Factors Regulating Absorption. 33

2.7.4  Iron Transport and Utilization. 35

2.7.5  Iron Requirements 36

2.7.6  Iron Deficiency (I.D) 36

2.7.7  Diagnosis of Iron deficiency. 37

2.7.8  Iron toxicity. 38

2.8    Calcium (Ca) 38

2.8.1  Dietary sources and Absorption of Ca. 39

2.8.2  Mechanism of Ca Absorption. 39

2.8.3  Factors Affecting Ca Absorption. 39

2.8.4  Regulation of Ca Concentration. 41

2.8.5  Functions of Ca. 42

2.8.6  Hypercalcaemia and Hypocalcaemia. 43

2.8.7  Measurement of total Ca. 43

2.9    Magnesium.. 44

2.9.1 Chemistry. 45

2.9.2  Dietary Sources 46

2.9.3  Absorption, Transport, Metabolism and Excretion of Magnesium.. 47

2.9.4  Functions Of Magnesium.. 50

2.9.5  Hypermagnesaemia. 51

2.9.5.1Causes of Hypermagnesaemia. 51

2.9.6  Hypomagnesaemia. 52

2.10  Effects of Malaria parasite on Micronutrients in Children. 53

2.11  Iron Levels in Malaria parasite Infection. 53

2.12  Calcium Levels in Malaria Parasite Infection. 54

2.13  Magnesium Levels in Malaria Parasite Infections 55

2.14  Folate Levels in Malaria Parasite Infection. 55

2.15  Vitamin B₁₂ Levels in Malaria Parasite Infection. 57

CHAPTER THREE. 58

3.0    MATERIAL AND METHODS. 58

3.1    Study Area. 58

3.2    Study Population. 58

3.3    Inclusion criteria. 58

3.4    Exclusion criteria. 58

3.5    Control group. 58

3.6    Sample Collection. 58

3.7    Laboratory Analysis 59

3.7.1  Procedure For Malaria Parasite Test 59

3.7.2  Assay Of Serum Calcium Concentration. 59

3.8    Quality Control 61

3.9    Analysis For Vitamim B12 And Folic Acid. 61

3.9.1  Electroluminescence (ECL) For Measurement of Vitamin B12. 61

3.9.2  Methodology/Steps 62

3.9.3  Enzyme-linked Immunosorbent Assay Kit For Folic Acid (FA) 63

3.9.4  Reagents and Quantity. 64

3.9.5  Methodology/Steps 64

3.9.6  Assay Of Serum Magnesium Concentration. 66

3.10  Procedure For Serum Iron. 68

3.11  Statistical Analysis 69

CHAPTER FOUR.. 70

4.0    RESULTS. 70

CHAPTER FIVE. 77

DISCUSSION.. 77

CONCLUSION AND RECOMMENDATION.. 80

REFERENCES. 81

LIST OF TABLES

Table 1: Food sources of Magnesium (Saris et al., 2000) 47

Table 2: Distribution of magnesium in the adult human. 49

Table 3: Procedure/protocol table for calcium estimation. 60

Table 4: Procedure/Protocol table for magnesium estimation. 67

Table 5: Age, sex, and malaria load of study and control subjects 70

Table 6: Comparison between the PCV, RBC, HgB conc., red cell indices, total and differential leucocyte counts, NLR, and PLR of the study and control groups. 73

Table 7: Comparing the calcium, magnesium, iron, vit. B12, and folic acid levels between the malaria Positive subjects and Malaria negative subjects 75

LIST OF FIGURES

Figure 1: Malaria cases (per 100,000) by Country Worldwide. 9

Figure 2: Life Cycle of Malaria Parasite (Ayodotun and Olugbenga, 2012). 12

Figure 3: Structure of Vitamin B₁₂ (Chatterjea and Shinde, 2012). 21

Figure 4: Mechanism of Absorption of Vit. B₁₂ (Chatterjea and Shinde, 24

Figure 5: Structure of Folacin. 27

Figure 6: Steps involved in the Formation of F.H4. 28

Figure 7: Structure of F.H4. 29

Figure 8: Absorption of Iron. 35

Figure 9: Electroluminescence method, competitive principle for measuring vitamin B12. 63

Figure 10: showing the ages of the study and control groups 71

Figure 11: showing the sex of the study and control groups 71

Figure 12: Comparison between the PCV, RBC, HgB conc., and red cell indices, of the study and control groups. 74

Figure 13: Comparing the calcium, magnesium, iron, Vit. B12, and folic acid levels between the malaria Positive subjects and Malaria negative subjects 76

 CHAPTER ONE

INTRODUCTION

1.1     Background of Study

Malaria is a prevalent disease in tropical and subtropical areas of Africa. It is estimated that 1-3 million deaths occurs worldwide, mostly involving children under the age of 5years (Gouado et al., 2007). Malaria is a major public health challenge in Nigeria and it accounts for more cases and deaths than any other country in the world (Olasehinde et al., 2010). This disease is often linked to changes in climate, poverty, malnutrition and the double resistance of the malaria parasite to usual anti-malaria drugs and insecticides (Müller and Garenne, 1999). Infection by malaria can cause serious health problems and this often leads to death especially in children (Gouado et al., 2007). The disease is caused by malaria parasites (Plasmodium species) which are transmitted by the female anopheles mosquito (vector). There are today more than 25 named plasmodium species which infect primates. Four of the species are human parasites; P. falciparum, P. vivax, P. malaria and P. ovale (Trampuz et al., 2003). Epidemiological studies have demonstrated that P. falciparum is the most dangerous specie as it is responsible for most of the deaths caused by malaria (Greenwood et al., 2005). The malaria parasite is transmitted when an individual is bitten by infected female anopheles mosquito (Ochei and Kolhatkar, 2008). The main symptom of uncomplicated malaria in children is fever. Older children may present with headache, backache, chills, myalgia and fatigue (Olasehinde et al., 2010). Severe anaemia may exist alone or in combination with other complications particularly cerebral malaria and respiratory distress in which it portends worse prognosis (WHO, 2004). Consequences of severe malaria include coma and death if untreated, young children are especially vulnerable (Anemana et al., 2004). Laboratory diagnosis of malaria could be made by detection of parasite in blood] or by serological techniques (Ochei and Kolhatkar, 2008).

Micronutrients are trace elements that are required in small quantities to ensure normal metabolism, growth and physical well‐being. Some studies relating micronutrient status and malaria infection reported low plasma levels of certain micronutrient in acute malaria infection (Alonso, 2004). Iron and beta carotene which are reported to have modulatory effect on the pathogenesis of malaria, have been observed to be deficient in acute plasmodium falciparum infection (Lavender, 1993; Shankar and Prasad 1998;; Beard, 2001; Caulfield et al., 2004). The levels of micronutrients in children are of particular interest since adequate intake is of great importance for the well being, proper development, and functioning of the body starting from fetal life and throughout childhood. Micronutrients comprise of vitamins and minerals. Examples of vitamins are vitamin A, pro-vitamin A (Beta‐carotene), vitamin B₁, vitamin B₂, vitamin B₆, vitamin B₁₂, biotin, vitamin C, vitamin E, vitamin D, vitamin K ,folic acid, niacin and pantothenic acid while minerals include the trace elements such as iron, copper, iodine, manganese, selenium and zinc together with the macro elements calcium, magnesium, potassium and sodium (Asaolu and Igbaakin, 2009; Crook, 2012). Micronutrients have been implicated to play important roles in immunity and physiologic functions. For instance, Calcium is an important nutrient that plays a major role in bone and teeth formation, impulse transmission, catalytic activation among others (Nordin, 1997). Iron plays an important role in the production of heamoglobin, oxygenation of red blood cells and lymphocytes. It improves the function of enzymes in protein metabolism and enhances the function of calcium and copper (Asaolu and Igbaakin, 2009). Vitamin B₁₂ is involved in the maturation of red blood cells. The folic acid coenzymes are specifically concerned with metabolic reactions involving the transfer and utilization of the one carbon moiety       (Crook, 2012). Micronutrients are found in small quantities within the body and they are obtained from a wide variety of foods.  No single food contains all of the micronutrients we need and, therefore, a balanced and varied diet is necessary for an adequate intake. Micronutrients deficiency is more frequent amongst children in developing countries (Gibson and Ferguson, 1998). These deficiencies may contribute to an increased risk of parasitic infection such as malaria (Mahomed, 2000).

1.2     Justification of Study

Malaria has been a major challenge to the world’s population especially in Africa and indeed Nigeria. It has been implicated in increased rate of morbidity and mortality among children (Sachs and Malaney, 2002).  Research has shown that malaria causes 0.5 – 3.0 million deaths each year and that 75% of these deaths occur in African children under the age of 5years (Greenwood et al., 2005).The increased clinical state of malaria infection may be due to poor nutritional status more especially as a result of micronutrients deficiency (Gouado et al., 2007). Micronutrients play vital role both in combating anaemia and other adverse effects of malaria infection in humans and animals in developing resistance against the disease. Micronutrients are not only necessary in the regeneration of heamolyzed red cells during malaria infection, but also served as antioxidants hence protecting the red cells against damage by malaria toxins (Jain, 2006). It is therefore of tremendous importance to assess micronutrients status of children with malaria.

1.3     Aim of Study

 The aim of this study is to assess the levels of micronutrients in children with malaria infection in Paediatric Ward, Central Hospital, Benin city.

1.4     Specific Objective

1.     To evaluate the levels of vitamin B­­_12, folic acid, iron, magnesium, and calcium in malaria infected children.

2.     To correlate the micronutrient levels with the severity of malaria infection.

3.     and to compare the results with  the control group  (non-infected children).

1.5     Research Design

This is a case study designed to assess micronutrients status of malaria infected children and then compare findings with non-infected children in Benin City.

1.6     Ethical consideration

Ethical approval was sought and approved by the ethical committee of Central Hospital, Benin City, Edo State.

1.7     Research Hypothesis

H₁:  malaria parasites affects calcium, magnesium, iron ,vitamin B_12,folic acid in children infected with malaria.

H₀ malaria parasites does not affect calcium, magnesium, iron ,vitamin B_12,folic acid in children infected with malaria.

1.8     Questionnaire/informed consent

A questionnaire, written in English was administered to the parents of the subjects to fill. An informed consent was given to the parents or guardian of the children before sample was collected.

1.9     Sample size

The sample size (N) was calculated  using prevalence from previous studies

The sample (Olasehinde et al., 2010). size for this study will be obtained using the formula described by Dean et al., (1995).

          N       =       Z²P (1 – P)

                                   D²

N       =       required sample size

Z       =       confidence level at 95% (standard value of 1.96)

P       =       estimated prevalence of intestinal parasites of pupils (84.7%)

D       =       margin of error at 5% (standard value = 0.05)

N=1.96² X 0.847(1-0.847)

0.05²

3.8416 X 0.847(0.153) 
   ___________________

0.0025

                N=199 minimum sample sizes

Therefore a minimum of 200 test samples and 100 control will be used for this research.

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