ASSESSMENT OF THE LEVELS OF LACTATE DEHYDROGENASES IN MALARIA INFECTION AND ITS EFFECTS ON ACRIDINE ORANGE STAINING DURING DIAGNOSIS IN PATIENTS FROM NGWO, UDI LOCAL GOVERNMENT AREA IN ENUGU STATE

ABSTRACT

Malaria is a potential medical emergency and should be treated accordingly. Delays in diagnosis and treatment are leading causes of death in many countries. The availability of rapid, simple diagnostic tools to substitute microscopy in malaria diagnosis will assist in the control of malaria by allowing therapy to be accurately administered. Emphasis has, recently been placed on alternative methods such as the use of acridine orange in malaria diagnosis using conventional fluorescence microscope or an interference filter system, but these were not considered a useful tool in field conditions due to weak illumination or high cost of the microscopes. This was the first study to use acridine orange in terms of their visual colours for malaria diagnosis. With acridine colour change, the infected individuals showed bright greenish yellow while the uninfected showed deep greenish yellow. Further trial was also made on the use of LDH substrate in combination with acridine solution based on colour changes, with the infected indicating bright brownish orange while the uninfected showed deep brownish orange. Thick and thin blood smears of Giemsa stain was used to estimate the parasite density and to determine the specific specie (Plasmodium falciparum). The evaluation of lactate dehydrogenase levels and concerntration of the study population showed mean values of 125.70±90.27unit/l and 0.0013±0.001mmol/l respectively. Though malaria infected individuals had higher LDH levels and concentrations than the uninfected individuals there was still no significant difference (p > 0.05) in the mean LDH levels and concentrations of infected and uninfected individuals. Statistically, of 100 individuals sampled, the prevalence according to the various methods were 65% for microscopy, 49% for acridine colour change and 51% for the combination (LDH substrate+ acridine solution). There was stronger agreement between the combinations with microscopy than in the acridine-microscopy relation. Generally, the sensitivity and NPV (Negative Predictive Value) for acridine colour change-microscopy were 75.38% and 68.63% respectively with 100% specificity PPV (Positive Predictive Value) while the sensitivity and NPV for the combination-microscopy relation were 78.46% and 71.43% with excellent specificity and PPV. Based on these findings, the study suggested that the use of these new methods could serve as alternatives to microscopy especially in most local setting where microscopy or trained technicians are unavailable.

TABLE OF CONTENTS

Title page

Abstract

Table of contents

List of figures

List of tables

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW

1.1       Introduction

1.1.1    Justification of the study

1.1.2    Objectives of the study

1.2       Literature Review

1.2.1    Human malaria

1.2.2    Signs and symptom of malaria

1.2.3    Diagnosis of malaria

1.2.3.1 Routine laboratory diagnosis

1.2.3.2 Microscopic diagnosis using stained thin and thick peripheral blood smears (PBS)

1.2.3.3 Thick blood film

1.2.3.4 Thin blood film

1.2.3.5 Sensitivity of thick blood film

1.2.3.6 Estimation of parasitemia by using blood films

1.2.3.7 Examination of thick blood films

1.2.3.8 Examination of thin blood films

1.2.3.9 Diagnosis using fluorescence microscopy

1.2.3.10 Detecting specific nucleic-acid sequences

1.2.3.11 Rapid diagnostic tests (RDTs)

1.2.3.12 HRP-2 immunochromatography-based assays

1.2.3.13 pLDH-Based immunochromatographic assays

1.2.3.14 Aldolase based immunochromatographic assays

1.2.3.15 Serological tests

1.2.3.16 Molecular diagnostic methods

1.2.3.17 PCR technique

1.2.3.18 LAMP technique

1.2.3.19 Microarrays

1.2.3.20 FCM assay

1.2.3.21 Automated blood cell counters (ACC)

1.2.3.22 Mass spectrophotometry

1.2.4    Life cycle of malaria

1.2.5    Pathogenesis of malaria

1.2.6    Malarial induced pathology (Hepatomegaly)

1.2.7    Prevention of malaria

1.2.7.1 Vector control of malaria

1.2.8    Treatment of malaria

1.3       Biochemistry and Physiology of Lactate Dehydrogenase

1.3.1    Lactate dehydrogenase

1.3.2    Catalytic function of LDH

1.3.3    Enzyme isoforms

1.3.4    Genetics in humans

1.3.5    Sources of LDH

1.3.6    LDH structure

1.3.7    LDH applications

1.3.8    LDH in clinical biochemistry

1.3.9    LDH in industrial processes

1.3.10  LDH in nanotoxicology

1.4       Development of Acridine Orange Fluorescence Microscopy

CHAPTER TWO: MATERIALS AND METHODS

2.1       Study Area

2.2       Ethical Clearance

2.3       Experimental Design

2.3.1    Sampling size, sampling method and sampling site

2.3.2    Collection and analysis of blood samples

2.3.3    Assay of LDH enzyme activity

2.3.4    Acridine orange effects experiment

2.4       Statistical Analysis

CHAPTER THREE: RESULTS

3.1       Characteristics of Study Population

3.2       Prevalence of Malaria by Microscopy

3.3       Parasite Density in Relation to Sex

3.4       Lactate Dehydrogenase (LDH) Levels in the Study Population

3.5       Lactate Dehydrogenase Levels in Males and Females in the Study Population

3.6       Parasite Densities and LDH Levels

3.7       Prevalence of Malaria based on Acridine Colour Change

3.8       Comparison of Colorimetric Reading of Malaria Infected and

            Uninfected based on Acridine

3.9       Reliability of Acridine Colour Change in the Detection of Malaria

3.10     Reliability of Acridine Colour Change in the Detection of Malaria

            based on Sex

3.11     Reliability of Acridine Colour Change in the Detection of Malaria

            Parasite based on Parasite Density

3.12     Diagnostic Performance of Acridine in Detection of Malaria

3.13     Prevalence of Combination of Acridine Orange and LDH in

            Detection of Malaria

3.14     Reliability of LDH + Acridine Test in Relation to Microscopy

3.15     Reliability of LDH + Acridine Test in Sex Detection of Malaria

3.16     Reliability of LDH + Acridine Test in Detection of Different

            Parasite Density

3.17     Sensitivity, Specificity, Predictive Values of LDH+Acridine as

            Rapid Diagnostic Test

3.18     Comparison of LDH + Acridine Diagnostic Performance with

            Acridine Only

3.19     Comparison of Acridine Performance and LDH + Acridine in

            Relation to Sex

3.20     Sensitivity, Specificity, Predictive Values of LDH+Acridine

            Compared with Acridine Only

CHAPTER FOUR: DISCUSSION, RECOMMENDATION AND CONCLUSION

REFERENCES

APPENDICES

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1        Introduction

Malaria is a mosquito-borne infectious disease of humans and other animals caused by parasite (a type of microorganism) of the genus Plasmodium (Collins, 2012). infection begins with a bite from an infected female mosquito (Anopheles Mosquito), which introduces the protists via its saliva into the circulatory system, and ultimately it reaches to the liver where they mature and reproduce (Sebastian et al., 2008). The life cycle of this parasite in the human host includes the developmental cycle in red blood cells, and the cycle taking place in the liver cell parenchyma, including a series of transformations in the host hepatocytes (Flornes, 2010). Pathophysiological processes usually associated with acute P. falciparum malaria infections, i.e., the hepatic activity of the invading sporozoites leading to centrilobular liver damage and the destruction of the host red blood cells consequent to erythrocytic merogony (Maegraith, 1981). The disease causes symptoms that typically include fever, anaemia and headache, which in severe cases can progress to coma or death (Fairhurst and Wellems, 2010). Five different species: P. falciparum, P. malariae, P. ovale, P. vivax, P.knowlesi, affect humans (Mueller et al., 2007; Collins, 2012). Plasmodium falciparum is the most pathogenic species and may cause severe malaria and death in untreated non-immume individuals (WHO, 2009). Disease transmission can be reduced by preventing mosquito bites by distribution of mosquito nets and insect repellents, or with mosquito control measures such as spraying insecticides and drain standing waters (Lengeler, 2004). Malaria continues to be one of the world’s most significant health problems, accounting for 300–500 million clinical attacks and over 1 million deaths every year mainly in children under 5 years of age (WHO, 1999). Although malaria remains a considerable public-health threat in parts of Asia and South America, 90% of its current burden is concentrated in sub-Saharan Africa (Carter, 2003). In areas where transmission is intense, malaria creates an array of biological and behavioural responses with long term impacts on social and economic growth and development (Sachs and Malaney, 2002). The growing recognition of the extent and impact of malaria on socio-economic development has prompted calls for greater investments, with an overall goal of improving health, particularly among the poor (Jha et al., 2002). Effective case management depends on both accurate and rapid diagnosis. A fast and easy to use method with high performance is required to differentiate malaria from non – malaria fevers (Vink et al., 2013). In the case of the falciparum malaria, delayed diagnosis may result in fatal outcomes (Rickman et al., 1989). More sensitive and timely methods other than Giemsa stain microscopy are needed, both for parasite detection and its levels of parasitaemae, by advancing in the use of other rapid diagnostic tests like lactate dehydrogenase, histindine rich protein II, acridine orange etc (Hanscheid, 1999).

Lactate dehydrogenase (LDH) is an intracellular enzyme, which catalyses the readily reversible reaction involving the oxidation of lactate to pyruvate with nicotinamide adenine dinucleotide (NAD) serving as coenzyme (Stryer, 1982). Plasmodium lactate dehydrogenase (PLDH) is a soluble glycolytic enzymes produced by the asexual and the sexual stages of the live parasites and it is released from the parasite infected erythrocytes (Kakkilaya, 2003). PLDH does not persist in the blood but clears about the same time as the parasites following successful treatment. The lack of antigen persistence after treatment makes the pLDH test useful in predicting treatment failure (Iqbal et al., 2004). LDH from P. vivax, P.malariae and P.ovale exhibit 90-92% identity to pLDH from P.falciparum (Brown et al., 2004). LDH is an enzyme, which is classified as a true intracellular Enzyme (Sullivan and Alpers, 1971), because of its high degree of tissue specificity. Overall tissue concentrations are some 500-fold greater than serum levels under normal circumstances (Podlasek and Mcpherson, 1989). LDH have five possible forms, which are found in human tissues e.g. liver, heart.....

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