ASSESSMENT OF MICRONUTRIENTS IN CHILDREN WITH MALARIA INFECTION
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 Vitamin 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
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 B12, 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 B12,folic acid in children infected with malaria.
H0 malaria parasites does not affect calcium, magnesium, iron ,vitamin B12,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.
.