DEVELOPMENT OF A RAPID URINE-BASED DIPSTICK TEST FOR DIAGNOSIS OF MALARIA BY URI SELORM MARKAKPO THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFULLMENT OF THE REQUIREMENT FOR THE AWARD OF PHD PUBLIC HEALTH DEGREE DEPARTMENT OF BIOLOGICAL, ENVIRONMENTAL AND OCCUPATIONAL HEALTH SCIENCES SCHOOL OF PUBLIC HEALTH, COLLEGE OF HEALTH SCIENCES UNIVERSITY OF GHANA, LEGON JULY, 2014 University of Ghana http://ugspace.ug.edu.gh i DECLARATION I hereby declare that this thesis is the result of my own original research, except for areas where specific references have been made and duly acknowledged. I also affirm that the studies reported in this document were carried out by me under the supervision of my team of academic advisors. Lastly, I declare that this work has not been submitted, either in part or in whole, to any other institution for an award of a degree. Uri Selorm Markakpo (M.Phil.) (10156003) Prof. Isabella A Quakyi (Ph.D.) (SUPERVISOR) Prof. Kwabena M. Bosompem (Ph.D.) (SUPERVISOR) Dr. Mawuli Dzodzomenyo (Ph.D.) (SUPERVISOR) University of Ghana http://ugspace.ug.edu.gh ii DEDICATION This thesis is dedicated to my supervisors, my children and my wife for the moral and emotional support given me during the trying moments of the conduct of the studies herein. University of Ghana http://ugspace.ug.edu.gh iii ACKNOWLEDGEMENT First, and foremost, I wish to express my sincere thanks to Professor Ernest Aryeetey, the current Vice Chancellor of University of Ghana (UG), Prof. Alexander K. Nyarko, the former Director of the Noguchi Memorial Institute for Medical Research (NMIMR), UG, and Prof. Richard Adanu, the current Dean of SPH, UG, for the various roles played towards the successful completion of this study. Secondly, I sincerely appreciate the efforts made by my supervisors, Prof. Isabella A. Quakyi, SPH, Prof. Kwabena M. Bosompem, NMIMR and Dr. Mawuli Dzodzomenyo, SPH, University of Ghana as well as my collaborating mentor, Prof. David Sullivan Jr. of the Malaria Research Institute, Department of Molecular Immunology and Microbiology, Johns Hopkins University School of Public Health, Baltimore, Maryland, USA, for their guidance and support towards the conduct of this study. I also like to thank Dr. William K. Anyan and Dr. Irene Ayi of the Parasitology Department, NMIMR, Dr. Michael F. Ofori and Prof. Ben Gyan of the Immunology Department, NMIMR, Mr. Alfred Dodoo of the Electron Microscopy and Histopathology Department, NMIMR and Dr. Daniel Boamah of the Parasitology Department, NMIMR and Centre for Scientific Research into Plant Medicine (CSRPM), Mampong, for the great contributions made towards the success of this thesis project. Furthermore, I like to express my deep appreciation to the Bill and Melinda Gates Foundation’ Grand Challenges Explorations, World Health Organization (WHO), the University of Ghana, the Johns Hopkins University School of Public Health and Prof. David Sullivan Jr for providing funds and/ or other logistic support required for the execution of this project. In addition, I am greatly indebted to the NMIMR and SPH all of the University of Ghana, Legon, and the Kpone Health Centre (KHC), Kpone-on-Sea, for providing me with the laboratory facilities where all the experiments involved in this thesis project were conducted. Also, my heartfelt gratitude goes to Dr. Mawuli Dzodzomenyo, and Prof. Julius Fobil, the past University of Ghana http://ugspace.ug.edu.gh iv and current Heads of the Department of Biological, Environmental and Occupational Health Sciences (BEOHS), SPH, and Dr. Mawuli K. Gyakobo, Dr. Isaac Anim-Baidoo and Mr. Richard H. Asmah for the encouragement, companionship and moral support given me during the course of this project work. My special appreciation also goes to Mrs. Naa Adjeley Frempong, Mr. Joseph Otchere, Mr. Kwadwo K. Frempong and the late Mr. Osei A. Duah of the Department of Parasitology, NMIMR, Mr. Jonas Danquah of the Department of Biochemistry, Cell and Molecular Biology, UG, and Mr. Parnor Madjitey of the Department of BEOHS, SPH as well as my field Assistants Mr. Isaac Ankrah and Mr. Nicholas Amoah for the assistance given me during the collection of samples and the conduct of the experiments leading to the production of this thesis. The great assistance in data entry given me by Ms. Tina Ayeh of the Department of Epidemiology, NMIMR, Ms. Philomena Amankwah of the Kpone Malaria Project and Mr. Oliver K. Adotey of the Madina Central SDA Church, Madina, is also highly appreciated. I am most grateful to Dr. Phyllis Addo and Dr. Samuel Adjei of the Animal Experimentations Department, NMIMR, for providing the facilities for the immunization experiments; the staff of Parasitology Department, NMIMR, particularly, Messrs. Jonas. R. K. Asigbee (deceased) and Joseph. Quartey, for their technical and moral support during the conduct of this research project; the Administrative Staff of SPH, especially, Ms. Hannah T. Addo of the Department of BEOHS; Staff of the College of Health Sciences, UG; my study participants and the Kpone study community as a whole for their contributions during the conduct of this research. Ultimately, I thank the Lord God Almighty for the protection, support and special insights He provided me to ensure the successful completion of this research project. University of Ghana http://ugspace.ug.edu.gh v TABLE OF CONTENTS CONTENTS PAGE DECLARATION ................................................................................................................ i DEDICATION................................................................................................................... ii ACKNOWLEDGEMENT ................................................................................................ iii TABLE OF CONTENTS .................................................................................................. v LIST OF TABLES........................................................................................................... xii LIST OF FIGURES ........................................................................................................ xiii LIST OF PLATES ........................................................................................................... xv ABBREVIATIONS ........................................................................................................ xvi SUMMARY.................................................................................................................... xix CHAPTER ONE ................................................................................................................ 1 1.0 GENERAL INTRODUCTION .................................................................................. 1 1.1 BACKGROUND AND RATIONALE ............................................................. 1 1.2 PROBLEM STATEMENT ............................................................................... 4 1.3 OBJECTIVES ................................................................................................... 6 1.3.1 Main Objective ........................................................................................................... 6 1.3.2 Specific Objectives..................................................................................................... 6 1.4 JUSTIFICATION ............................................................................................................ 7 CHAPTER TWO ............................................................................................................... 8 LITERATURE REVIEW .................................................................................................. 8 2.1.0 MALARIA ........................................................................................................ 8 2.1.1 Economic Cost of Malaria ......................................................................................... 8 2.1.2 Geographic Distribution of Malaria and the Plasmodium Parasites .......................... 9 2.1.3 The Biology and Life-Cycle of the Plasmodium Parasite ........................................ 13 2.1.4 Clinical Signs and Symptoms of Malaria ................................................................ 17 2.1.5 Pathogenesis of Malaria ........................................................................................... 18 2.1.6 Classification of the Plasmodium Parasite ............................................................... 21 2.2.0 ANTIBODIES ................................................................................................. 24 2.2.1 The Basic Structure of Antibodies ........................................................................... 24 2.2.2 Classification of Antibodies ..................................................................................... 28 University of Ghana http://ugspace.ug.edu.gh vi 2.2.3 Classification of Antibodies According to Multiplicity of Clones .......................... 31 2.2.4 Uses and Applications of Monoclonal and Polyclonal Antibodies .......................... 37 2.3.0 IMMUNIZATION OF LABORATORY ANIMALS ..................................... 40 2.3.1 Introduction .............................................................................................................. 40 2.3.2 Route of Immunization of Laboratory Animals for Antibody Production .............. 40 2.4.0 IMMUNITY TO MALARIA .......................................................................... 48 2.4.1 Innate Immunity to Malaria ..................................................................................... 48 2.4.2 Acquired Immunity to Malaria................................................................................. 49 2.4.3 Vaccines against Malaria-1 ...................................................................................... 50 2.5.0 DIAGNOSIS OF MALARIA ......................................................................... 52 2.5.1 Clinical Diagnosis of Malaria .................................................................................. 53 2.5.2 Microscopic Diagnosis of Malaria ........................................................................... 54 2.5.3 Rapid Diagnostic Tests for Malaria ......................................................................... 57 2.6.0 MALARIA TRANSMISSION AND ITS EFFECT ON AGE-DEPENDENT DISEASE BURDEN IN ENDEMIC POPULATIONS ......................... 58 2.6.1 Malaria Transmission at Kpone-on-Sea ................................................................... 59 CHARPTER 3 ................................................................................................................. 61 GENERAL MATERIALS AND METHODS ................................................................. 61 3.1.0 STUDY SITE .................................................................................................. 61 3.1.1 Kpone-on-Sea ........................................................................................................... 61 3.2.0 STUDY DESIGN ............................................................................................ 62 3.2.1 Sample Size Determination ...................................................................................... 62 3.2.2 In Vitro Propagation of Laboratory (3D7) Strain of Plasmodium falciparum Parasites ................................................................................................. 64 3.2.3 Extraction of Soluble Crude Plasmodium falciparum Antigens from In Vitro Cultured 3D7 Parasites ............................................................................................. 65 3.2.4 Preparation of Non-Immune Sera and Antisera of Mice for Microplate ELISA ..... 67 3.2.5 Coating of Microplates for ELISA ........................................................................... 67 3.2.6 Screening of BALB/c Mice for Anti-Plasmodium Antibodies by Indirect ELISA ......................................................................................................... 68 CHAPTER FOUR ........................................................................................................... 70 IDENTIFICATION OF MALARIA INFECTED AND UNINFECTED SUBJECTS IN AN ENDEMIC POPULATION ............................................................................... 70 University of Ghana http://ugspace.ug.edu.gh vii 4.1.1 Objectives ................................................................................................................. 71 4.2.0 MATERIALS AND METHODS .................................................................... 71 4.2.1 Study Subject Clinical History and Baseline Characteristics .................................. 71 4.2.2 Urine Sample Collection and Analysis .................................................................... 72 4.2.3 Blood samples collection and analysis ..................................................................... 72 4.3.0 RESULTS ....................................................................................................... 76 4.3.1 Characterization of Plasmodium falciparum infected subjects from a malaria infected population .......................................................................... 76 4.4.0 DISCUSSION ................................................................................................. 87 CHAPTER FIVE ............................................................................................................. 95 EXTRACTION AND CHARACTERIZATION OF BIOMARKERS OF MALARIA FROM INFECTED HUMAN URINE ......................................................... 95 5.1.0 INTRODUCTION (1) ..................................................................................... 95 5.1.1 Aim ................................................................................................................... 96 5.1.1 (b) Specific Objectives.................................................................................... 96 5.2.0 MATERIALS AND METHODS .................................................................... 96 5.2.1 Characterization of the Sample Population for the Studies to Characterize Biomarkers of Malaria from Infected Urine ............................................................ 96 5.2.2 Determination of Malaria Infection Status of Study Participants ............................ 97 5.2.3 Determination of Blood Haemoglobin Concentration and the Presence or Absence of Anaemia ............................................................................. 97 5.2.4 Extraction of Plasmodium Proteins from Malaria Positive Urine ........................... 97 5.2.5 Urine analysis by BioRad protein assay................................................................. 100 5.2.6 Test Principle for Detection of Plasmodium Antigens in Samples by Different Commercial Rapid Diagnostic kits:........................................................ 100 5.2.7 Detection of Plasmodium Antigens in Infected Urine, Blood and Cultured P. falciparum Parasites by Rapid Diagnostic Test .................................................... 101 5.2.8 Detection of Malaria Proteins in Infected Urinary and Cultured Antigen Extracts by SDS-PAGE ............................................................................ 102 5.2.9 Coomassie Brilliant Blue R-250 Staining of Gel ................................................... 103 5.2.10 Identification of Biomarkers of Malaria by Western blotting Analysis ................. 104 5.2.11 Characterization of urinary hepcidin from Plasmodium infected human urine ................................................................................................................. 105 University of Ghana http://ugspace.ug.edu.gh viii 5.3.0 RESULTS ..................................................................................................... 107 5.3.1 Detection of P. falciparum HRP2 Antigen in Infected Human Urine by Commercial Diagnostic Kits ................................................................... 107 5.3.2 Detection of Plasmodium LDH Antigens in Infected Human Urine by the CareStartTM Rapid Diagnostic Kit .......................................................................... 108 5.3.3 Urine analysis by BioRad protein assay................................................................. 108 5.3.4 Characterization of proteins in Different Urine and Plasmodium Antigens by SDS-PAGE ........................................................................................ 110 5.3.5 Plasmodium Proteins in Different Antigen Preparations by Western Blotting Analysis ................................................................................................................. 112 5.3.6 Characterization of Hepcidin from Plasmodium Infected Human Urine ............... 113 5.3.7 Association between urinary hepcidin level and parasitaemia, anaemia and haemoglobin level .................................................................................................. 114 5.4.0 DISCUSSION ............................................................................................... 117 CHAPTER SIX............................................................................................................. .124 GENERATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST PLASMODIUM falciparum ANTIGENS using URINARY AND in vitro CULTURED parasites ......................................................... 124 6.1.0 INTRODUCTION ........................................................................................ 124 6.1.1.0 Aim ................................................................................................................. 125 6.2.0 MATERIALS AND METHODS .................................................................. 125 6.2.1 Preparation of Immunogens for Immunization of BALB/c Mice .......................... 125 6.2.2 Immunization of BALB/c Mice ............................................................................. 126 6.2.3 Anti-Plasmodium parasite Antibodies in Immunized Mouse Sera by Microplate ELISA .................................................................................................. 128 6.2.4 Feeder Cell Preparation and In Vitro Propagation of Myeloma Cells ................... 128 6.2.5 Cell Fusion and Selection of Hybridomas ............................................................. 129 6.2.6 Screening, cloning and cryopreservation of hybridoma cells ................................ 131 6.2.7 Determination of cut-off point for ELISA negative reactivity results ................... 131 6.2.8 Immunoglobulin Classes of Monoclonal Antibodies in Hybridoma Culture Secretions ............................................................................................................... 132 6.3.0 RESULTS ..................................................................................................... 132 6.3.1 Immune Response in BALB/c Mice to Urinary and Cultured Plasmodium Antigens ................................................................................................................. 132 University of Ghana http://ugspace.ug.edu.gh ix 6.3.2 Detection of Anti-Urinary Plasmodium Antibodies in Immunized Mouse Sera by Microplate ELISA ......................................................................... 133 6.3.3 Detection of Cultured Plasmodium Parasite Antibodies in Immunized Mouse Sera by Indirect ELISA ........................................................... 135 6.3.4 Characterization of Anti-Plasmodium Monoclonal Antibodies by Microplate ELISA .................................................................................................. 139 6.3.5 Immunoglobulin Class and Reactivity of Monoclonal Antibodies by Rapid Isotyping Kit (PRIK) .............................................................................................. 140 6.3.6 Cloning of Hybridoma Cells the Limiting Dilution Method ................................. 141 6.4.0 Discussion ..................................................................................................... 142 CHAPTER SEVEN ....................................................................................................... 148 REACTIVITY OF MOUSE ANTI-PLASMODIUM MONOCLONAL AND POLYCLONAL ANTIBODIES IN ELISA AND WESTERN BLOTTING Assay ...................................................................................................................... 148 7.1.0 INTRODUCTION ........................................................................................ 148 7.1.1 Overall Aim ............................................................................................................ 149 7.2.0 MATERIALS AND METHODS .................................................................. 150 7.2.1 Optimum Conditions for ELISA ............................................................................ 150 7.2.2 Reactivity of Anti-Plasmodium Monoclonal Antibodies by Microplate ELISA ... 150 7.2.3 Prevalence of Malaria in the Sample Population by MAb Microplate ELISA ...... 151 7.2.4 Prevalence of Malaria in the Sample Population by anti-Plasmodium PAb Microplate ELISA .................................................................................................. 152 7.2.5 Reativity of Anti-Plasmodium PAbs to Different Antigen Preparations by Western Blotting Analysis ................................................................................ 152 7.2.6 Reactivity of UCP4W7 MAb to different antigen preparations by SDS-PAGE and western blotting assay-1 .............................................................. 153 7.2.7 Reactivity of UCP4W7 MAb to different antigen preparations by ............................. SDS-PAGE and western blotting assay-2 .............................................................. 154 7.3.0 RESULTS ..................................................................................................... 156 7.3.1 Optimum Conditions for ELISA to Ascertain the Reactivity of Anti- Plasmodium PAbs and MAbs ....................................................................... 156 7.3.2 Reactivity of mouse anti-Plasmodium MAbs to urinary and cultured parasite antigens by microplate ELISA-1 ............................................................................ 156 7.3.3 Reactivity of mouse anti-Plasmodium MAbs to urinary and University of Ghana http://ugspace.ug.edu.gh x cultured parasite antigens by microplate ELISA-2 ................................................ 159 7.3.4 Summary of the reactivity of mouse anti-Plasmodium MAbs to urinary and cultured parasite antigens ............................................................................... 161 7.3.5 Reactivity of UCP4W7 MAb to different antigen preparations by SDS-PAGE and western blotting assay-1 .............................................................. 162 7.3.6 Separation of proteins in different antigen preparations by SDS-PAGE and reactivity of UCP4W7 by western blotting assay-2 ........................................ 164 7.3.7 Detection of Plasmodium Proteins in Different Fractions of Urine and Plasmodium Antigens by Western Blotting Analysis ..................................... 167 7.3.8 Relationship between Molecular Weights of Standard Proteins and Their Mobility through the NuPAGE-MOPS Gel ................................................. 168 7.3.9 Molecular Weights of Standard Proteins and Their Mobility through the NuPAGE-MOPS Gel ....................................................................................... 169 7.3.10 Estimation of Molecular Weights (MW1) of Unknown Proteins .......................... 170 7.3.11 Relative sensitivity and specificity of tests for different biomarkers of malaria ................................................................................................................. 171 7.3.12 Relative sensitivity and specificity of UCP4W7 ELISA and quatitative buffy coat test for malaria ...................................................................................... 173 7.4.0 Discussion ..................................................................................................... 174 CHAPTER EIGHT ........................................................................................................ 183 GENERAL DISCUSSION AND CONCLUSIONS ..................................................... 183 REFERENCES .............................................................................................................. 198 APPENDICES ............................................................................................................... 224 APPENDIX 1: MAP OF KPONE-ON-SEA STUDY AREA ...................................... 224 APPENDIX 2: ADDITIONAL DATA FROM CHAPTER FOUR ............................. 225 APPENDIX 3: SCHEMATIC REPRESENTATION OF THE RAPID DIAGNOSTIC TEST PROCEDURE AS SHOWN IN WHO (2000) .......................... 230 APPENDIX 4: RESULTS OF URINE ANALYSIS .................................................... 231 APPENDIX 5: PUBLICATIONS .................................................................................. 247 Appendix 5a A poster presented at the First Annual College of Health Sciences Conference, held on 26th to 28th September, 2007 .................................. 247 Appendix 5b A publication on the relationship of parasitemia and anemia University of Ghana http://ugspace.ug.edu.gh xi among patients with Plasmodium falciparum malaria in Ghana ............ 248 APPENDIX 6: ETHICAL CONSENT FORMS ........................................................... 252 Appendix 6a ................................................................................................................. 252 Appendix 6b ................................................................................................................. 254 Appendix 6c ................................................................................................................. 256 APPENDIX 7: ETHICAL CLEARANCE CERTIFICATE .......................................... 257 APPENDIX 8: TECHNICAL CHALLENGES ENCOUNTERED IN THE COURSE OF THE STUDY AND HOW THEY WERE CIRCUMVENTED .............................................................................. 258 University of Ghana http://ugspace.ug.edu.gh xii LIST OF TABLES Table 1: Host and Geographic Distribution of some Plasmodium parasites .......................... 12 Table 2: Classification of antibodies based upon location, structure and function ................. 30 Table 3: Study Subject Clinical History and Baseline Characteristics ................................... 78 Table 4: Association between Malaria and Sex ...................................................................... 79 Table 5: Prevalence of Parasitaemia among Plasmodium Parasite Positive Subjects Relative to Age............................................................................................ 81 Table 6: Association Between Malaria and Anaemia ............................................................. 84 Table 7: Association between malaria and fever .................................................................... 87 Table 8: Characterization of Hepcidin from Plasmodium Infected Human Urine and Study Subjects Characteristics .............................................................. 114 Table 9: Association between urinary hepcidin level and parasitaemia, haemoglobin level and anaemia ............................................................................. 116 Table 10: Mode of grading of monoclonal antibody reactivity patterns ............................... 131 Table 11: Immunoglobulin classes of selected hybridoma culture secretions ...................... 140 Table 12: Reactivity of anti-Plasmodium MAbs to urinary and cultured parasite antigen extracts-1 .................................................................................... 158 Table 13: Reactivity of mouse anti-Plasmodium MAbs to urinary and cultured Plasmodium antigen extracts .................................................................. 160 Table 14: Summary of monoclonal antibody reactivity results ............................................ 161 Table 15: Relationship between the molecular weights of standard proteins and their mobility through NuPAGE MOPS gel .................................... 168 Table 16: Estimation of molecular weights (MW1) of unknown proteins ........................... 170 Table 17: Estimation of molecular weights (MW2) of unknown proteins ........................... 171 Table 18: Mean molecular weights of unknown proteins ..................................................... 171 Table 19: Sensitivity and specificity of tests for different biomarkers of malaria ................ 172 Table 20: Sensitivity and specificity of UCP4W7 ELISA and QBC tests ............................ 173 University of Ghana http://ugspace.ug.edu.gh xiii LIST OF FIGURES Figure 1: Global distribution of malaria (Sachs and Malaney, 2002) .............................. 11 Figure 2: General and Ultra-structure of the P. falciparum merozoite ............................ 14 Figure 3: Life cycle of the Plasmodium falciparum malaria parasite in the human and the mosquito hosts (Bannister and Sherman, 2009) ................. 15 Figure 4a and b: Basic structures of the immunoglobulin molecule ....................................... 28 Figure 5 Morphological views of the various stages of the four (4) main human malaria parasite species ............................................................... 73 Figure 6: Analysis of blood by hematocrit system for haemoglobin concentration ........ 75 Figure 7: Prevalence of Malaria among Study Subjects Relative to Age, Parasite Density and Grading ........................................................................... 80 Figure 8: Prevalence of malaria among study participants in relation to age .................. 82 Figure 9: Variation of Mean Parasite Density Among Malaria Positive Subjects in Relation to Age .............................................................................. 83 Figure 10: Variation of Mean body (Axillary) Temperature of Study Subjects in Relation to Malaria Infection and Age ........................................... 86 Figure 11: Flow chart for extraction of Plasmodium antigens from urine of infected individuals ............................................................................. 99 Figure 12: Determination of Concentration of Proteins in Plasmodium Infected Urine by BioRad Assay .................................................................... 109 Figure 13: Coommassie blue gel electrophoresis results ................................................. 111 Figure 14: Western blotting results .................................................................................. 112 Figure 15: Immune Response of BALB/c Mice to Urinary Malaria Antigens ............... 134 Figure 16: Immune Response in BABLB/c mice to Cultured Plasmodium Antigens ..................................................................................... 135 Figure 17: Reactivity of Anti- Plasmodium Monoclonal Antibodies by Microplate ELISA .......................................................................................... 139 Figure 18: Mode of arrangement of the western blotting apparatus and gel system for western blotting analysis ......................................................... 153 Figure 19: a) Analysis of proteins in different antigen preparations by SDS-PAGE-1 and b) UCP4W7 probed western blotting assay-1. ................... 163 Figure 20: a) Analysis of proteins in different antigen preparations by SDS-PAGE-2. and b): UCP4W7 probed western blotting assay-2. ............... 166 University of Ghana http://ugspace.ug.edu.gh xiv Figure 21: Western blotting results ....................................................................................... 167 Figure 22: Molecular weights of standard proteins and their mobility through the NuPAGE-MOPS Gel ....................................................................... 169 University of Ghana http://ugspace.ug.edu.gh xv LIST OF PLATES Plate 1: A commercial RDT cassette showing the presence of P. falciparum histidine rich protein II (pfHRP2) in crude antigens (PAgHU) extracted from malaria infected human urine ..................... 107 Plate 2: A commercial RDT cassette showing the presence of P. falciparum histidine .rich protein II (pfHRP2) in crude antigens (PAgHU) extracted from malaria infected human urine 108 Plate 3: Collecting blood from the tail of BALB/c mouse before and after immunization ..................................................................................................... 127 Plate 4: Inoculating BALB/c mouse with PAgHU or CPfAg Immunogen ..................... 127 Plate 5: A microtitre plate showing the response of mice 1 and 2 to urinary Plasmodium antigens as determined by microplate ELISA ............................... 136 Plate 6: A microtitre plate showing response of mice 3 & 4 to urinary Plasmodium antigens as determined by microplate ELISA:.............................. 137 Plate 7: A cell fusion product on day-3 post cell fusion, showing a well on a 24-well tissue culture plate containing a mixture of unfused myeloma cells, spleen cells and hybridoma cells resulting from cell fusion. .................................................................................. 138 Plate 8: Hybridoma cell colonies that are actively dividing and are in various phases of growth on day 9 post cell fusion.and 14 post cell fusion. ............................................................................................. 138 Plate 9: Hybridoma cell colonies that are actively dividing and are in various phases of growth on days 14 post cell fusion. ................................... 138 University of Ghana http://ugspace.ug.edu.gh xvi ABBREVIATIONS µg: Microgram µl: Microlitres µm: Micrometer ABTS: 2, 2-Azino-bis (3-ethylbenethiazoline-6-sulphonic acid) ACT: Ammonium chloride and tris (hydroxymethyl, aminomethane) BCE Before Common Era or, before the first period of the Gregorian calendar BSA: Bovine serum albumin C7H5O2Na: Sodium benzoate CBB Carbonate-bicarbonate buffer Co(NO3)2.6H2O: Cobalt nitrate, hexahydrate CPfAg Cultured Plasmodium falciparum antigen extract CPR Cytochrome P450 reductase CSA: Condroitin Sulfate A CW: Control wells DAB: 3, 3’-Diaminobenzidine DED: Dangme East District dH2O: Deionized water ddH2O: Double deionized water DMSO: Dimethyl sulfoxide DNA: Deoxyribonucleic acid ELISA: Enzyme-linked immunosorbent assay FBS: Fetal bovine serum g: Gram Ga: Gauge GPCI: Global Parasite Control Initiative GST: Glutathione S-transferase HA: Hyaluronic acid HBMM: Home-based management of malaria H2O2: Hydrogen peroxide HAT: Hypoxanthine aminopterin thymidine HGPRT: Hypoxanthine guanine phosphorybosyl transferase HIV: Human immunodeficiency virus HRP: Horseradish peroxidase PAgHU Human urinary Plasmodium parasite antigen extract ICAM Intracellular adhesion molecule ICT: Immuno-chromatographic Test IFA: Indirect immunofluorescent assay IgA: Immunoglobulin A IgE: Immunoglobulin E IgG: Immunoglobulin G IgM: Immunoglobulin M IHA: Indirect haemagglutination IMDM: Iscove’s modified Dulbecco’s medium JICA: Japan International Cooperation Agency KCl: Potassium chloride KHC: Kpone Health Centre KHPO4 Potassium hydrogen phosphate University of Ghana http://ugspace.ug.edu.gh xvii KMP: Kpone Malaria Project KOS: Kpone-on-Sea mA: Milliampere MSS Mass Spectrometry Sequencing mg: Milligram min: Minute ml: Millilitre mm: Millimeter mM: Millimolar MAb: Monoclonal antibody MBT Mean body temperature MPD Mean parasite density Mr: Molecular mass MW Molecular weight MWCO: Molecular weight cut off Na2HPO4: Disodium hydrogen phosphate NaCl: Sodium chloride NaCO3: Sodium carbonate Na2-EDTA: Disodium ethylene diamine tetra acetate NaHCO3: Sodium hydrogen carbonate (sodium bicarbonate) nm: Nanometer NMCP: National Malaria Control Programme NMIMR: Noguchi Memorial Institute for Medical Research PBS Phosphate buffered saline PDCD: Parasitic Diseases Control District PEG: Polyethylene glycol Pf (P. falciparum) Plasmodium falciparum pfEMP1: Plasmodium falciparum erythrocyte membrane protein 1 PRIK Pierce rapid isotyping kit PVDF: Polyvinylidene diflouride RBC: Red blood cell (erythrocyte) RDT Rapid diagnostic test RIA: Radioimmunoassay RT: Room temperature RUBDA Rapid urine-based dipstick assay SASS: Saturated ammonium sulphate solution SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis sec Second(s) SPH: School of Public Health SWA: Soluble worm antigens TBS: Tris-buffered saline TEMED: N, N, N’-N’-tetramethylethylenediamine Th1: T helper type 1 Th2: T helper type 2 TK: Thymidine kinase TW: Test wells RUBDA: Urine-Base Dipstick Test v/v: Volume per volume w/v: Weight per volume WACIPAC West African Centre for International Parasite control University of Ghana http://ugspace.ug.edu.gh xviii WHO: World Health Organization 3D7 Plasmodium falciparum clone 3D7 μg Microgramme μl Microliter μM Micromolar °C Celsius cm Centimeter Da Dalton kDa kilodalton ICAM Intercellular adhesion molecule IFN Interferon Ig Immunoglobulin MACS Magnetic activated cell sorter OD Optical density UCP4W7 Uri, culture plate-4, well-7 University of Ghana http://ugspace.ug.edu.gh xix SUMMARY Bloodsmear microscopy is currently the gold standard and the principal test for confirmatory diagnosis of malaria. However, microscopy is labour-intensive and is limited by inadequate sensitivity and specificity. Apart from microscopy, rapid diagnostic tests (RDTs), which include the quantitative buffy coat test (QBC) and the immuno-chromatographic tests (ICTs) are alternative tests for confirmatory diagnosis of malaria. RDTs, particularly, ICTs are fast gaining momentum as alternative tests for routine diagnosis of the disease. These RDTs are fast and have yielded remarkable diagnostic outputs in certain localities, however, they are invasive, and sometimes, have limited sensitivity and/ or specificity. The work reported in this thesis was performed in order to generate monoclonal antibodies (MAb) towards the development of rapid urine-based assay (RUBDA) for non- invasive diagnosis of malaria, through detection of parasite antigens in the urine of infected individuals. Such an antibody-based test, is hoped to serve as a promising alternative for diagnosis of the disease, especially, for home-based management of malaria, particularly, in the hard-to-reach, low-income communities where the morbidity and mortality are highest. Urine and blood samples were collected from Kpone-on-Sea (KOS), a malaria endemic coastal fishing village in south-eastern Ghana. KOS is being developed as a model site for malaria intervention studies in a larger study titled the Kpone Malaria Project (KMP). Blood smear microscopy, quantitative buffy coat (QBC) test and commercial rapid diagnostic test (RDT) kits were used to distinguish between malaria infected and uninfected blood and urine samples. This was necessary, partly to select infected urinary antigens for immunizations towards MAb generation. Also, it was necessary to select true negative control urine samples for ascertaining the specificity of the MAb. University of Ghana http://ugspace.ug.edu.gh xx Analysis of the results showed a high malaria prevalence of 46.5% among the study population. Also, the results showed that malaria was significantly associated with sex (P = 0.0001), age (P= 0.0001), and fever (P = 0.0001). The results however showed that malaria was not associated with anaemia (P = 0.173). The observation of significantly higher prevalence of malaria and higher parasite density among children than adults in the studies were important findings that confirmed reports by other writers that children are more susceptible to the disease than adults. In addition, the occurrence of higher prevalence of malaria among females in this community, even though it confirmed what Ayele et al. (2012) reported, was suspected to be linked to behaviour more than to sex, as reported by other authors (Steketee et al., 2001). Immunological and biochemical analyses needed to ascertain the link between malaria and sex were not carried out so it was difficult to conclude that malaria was sex-linked. Immunization of mice towards MAb generation showed that 30 µg of urinary antigen was more immunogenic than 15 µg. Mice immunized with 15 µg of the antigen showed weak immune response, with absorbance values ranging between 0.36 and 0.45, measured at 414 nm. A majority (80%) of these mice did not show any observable response at all. In contrast, almost all (93.3%) of the 15 mice immunized with 30 µg of immunogen, showed high immune response (absorbance = 2.75) with only one (6.7%) not showing any response at all. Also, the Plasmodium-infected human urinary antigen extract (PAgHU) was found to be more immunogenic than the cultured parasite antigen extract (CPfAg) in these experiments. In all, 96 MAb clones were successfully generated against urinary and cultured Plasmodium parasite antigens. Determination of the immunoglobulin class of some of the MAb clones produced showed that 57.14% were of the IgM and 42.86% were of the IgG isotype. Characterization of these MAbs by microplate ELISA showed that they were all (100%) reactive to urinary Plasmodium parasite antigen extract (PAgHU). Also, 66 (68.8%) University of Ghana http://ugspace.ug.edu.gh xxi of these clones were reactive to cultured parasite antigens (CPfAg), 30 (31.3%) reacted specifically to only PAgHU, and none reacted with CPfAg alone. One of the MAb clones (UCP4W7), showed strong reactivity in ELISA and immunoglobulin class analysis. This UCP4W7 MAb was consequently selected and evaluated by microplate ELISA for sensitivity and specificity in detecting malaria antigens in the urine of a cohort of 420 randomly selected individuals from the study community. In this experiment, using microscopy as a gold standard test, the UCP4W7 MAb distinguished between infected and uninfected urine with a relative sentivity and specificity of 96.9 and 75.6%, respectively. Compared to the UCP4W7 MAb, the mouse anti-Plasmodium PAb even though it had a relatively higher sensitivity (97.4%) in microplate ELISA the specificity was extremely lower (21.3%). Also, determination of the relative sensitivities and specificities of the tests for various biomarkers of malaria compared to microscopy showed that tests for ketones, microhematuria, leukocytes, nitrites, glucose, bilirubinuria urobilinogen as well as PAb ELISA, were highly specific but had very low sensitivities. Each of these tests had a combined reactivity (Relative sensitivity + specificity) being less than the 170% threshold required to qualify it as a promising biomarker for diagnosis of malaria. Characterization of proteins in PAgHU, CPfAg and other antigen preparations by SDS- PAGE showed that PAgHU contained more protein bands in the profile than the rest of the antigen preparations. The results also showed that the number of bands in the profiles of various urine samples from infected individuals decreased with decreasing parasite density. These findings demonstrated the likelihood of association between proteinuria and parasite density, and therefore, suggested that perhaps resolution of urinary Plasmodium proteins by SDS-PAGE could be used to quantify parasite density in infected individuals. University of Ghana http://ugspace.ug.edu.gh xxii Further characterization of the urinary malarial antigens specific to UCP4W7, using microplate ELISA, SDS-PAGE, western blotting assay, mass spectrometry sequencing (MSS) and commercial RDT kits, showed that some of these antigens comprised the P. falciparum species-specific histidine-reach protein 2 (HRP2), Plasmodium pan-specific lactate dehydrogenase (LDH) antigen, the anti-malaria human metabolite peptide hormone (hepcidin), as well as other malarial proteins whose identities are yet to be determined. The western blotting assay also revealed that the selected UCP4W7 MAb could distinguish between Plasmodium infected and uninfected human urine. In addition, it was found that the MAb could differentiate between urinary and cultured Plasmodium parasite antigens by western blotting assay. Furthermore, the western blotting analysis demonstrated that UCP4W7 could distinguish specifically between urinary Plasmodium antigens and antigens from Schistosoma parasites as well as poliomyelitis and measles vaccines. These results showed that species- specific MAbs against Plasmodium parasite proteins in urine would be promising for diagnosis of malaria and should therefore be explored further for development of accurate non-invasive tests for diagnosis of the disease. The results, however, showed that the UCP4W7 MAb could not distinguish between malaria antigens in urine and yellow fever vaccine antigens because of non-specific reactivity. On the other hand, cross-reactivity of the MAbs with yellow fever vaccine suggests that the Plasmodium-infected urine samples used in generating the MAbs, also had yellow fever antigens possibly originating from vaccination and/ or active infections with the virus. In this respect, it was thought that ruling out yellow fever vaccination or infections from malaria suspected urine samples, would enable the UCP4W7 MAb to be used in its current state to distinguish between malaria infected and uninfected persons. University of Ghana http://ugspace.ug.edu.gh xxiii Finally, examination of urine samples from malaria infected and uninfected individuals by MSS, demonstrated that urinary hepcidin was associated with malaria parasitaemia in infected individuals, but was not associated with anaemia. Hepcidin is a peptide hormone produced by chronic inflammation in affected individuals. Since malaria causes inflammation in infected persons, the results were thought to suggest that urinary hepcidin would be a promising biomarker for malaria. Ultimately, these results therefore suggested that urinary hepcidin could be used to develop a non-invasive test for diagnosis of malaria. University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE 1.0 GENERAL INTRODUCTION 1.1 BACKGROUND AND RATIONALE Malaria is a global health problem caused by protozoan parasites of the genus Plasmodium. It exerts a heavy toll of illness and death (WHO fact sheet 2002). Four main species of Plasmodium were originally known to infect humans, namely, P. falciparum, P. malariae, P. vivax, and P. ovale, of which, P. falciparum is the most predominant and lethal (WHO fact sheet 2002; White, 1998; Bradley, 1996). However, currently, P. knowlesi which was originally known to parasitize macaque monkeys of Southeast Asia has been reported to infect humans by many Authors (Sermwittayawong et al., 2012; Van den Eede et al., 2009). According to WHO, in 2013, about 198 million people were infected with malaria globally, 384,000 people died from the disease, 3.2 billion people, constituting about 40% of the world population was at risk of the disease (WHO 2014). It is sad to note however that albeit so much is being done to control the disease, malaria is still a major global health problem. This situation was corroborated by WHO World Malaria Report, 2008, which reported that in 2006, approximately 3.3 billion people were at risk of developing malaria each year, with at least 500 million cases, and a death toll of nearly 1.0 million annually. Sub- Saharan Africa is most affected (Snow et al., 2005), where malaria accounts for up to a third of all hospital admissions (Phillips, 2001; WHO fact sheet 2002). Children and pregnant women are at the greatest risk, due to lack of adequate immune protection against the disease (Schwartlander, 1997; WHO, 1996). In areas where Plasmodium falciparum is endemic, delay in treatment of uncomplicated malaria may result in severe disease in these most vulnerable groups, characterized by conditions such as, anaemia, weakness, cerebral malaria, obstetric complications and spontaneous abortion in University of Ghana http://ugspace.ug.edu.gh 2 pregnancy malaria, impaired consciousness, and death (Brewster et al., 1990; WHO fact sheet 2002). Early diagnosis and treatment are therefore crucial in preventing complicated malaria. In Ghana, malaria due to P. falciparum is a major cause of morbidity and mortality (Quakyi, 1980). It accounts for 42% of all outpatient clinic visits, 8% of all certified deaths and 20% of under-five mortality (GHS/MOH, 2004.). Furthermore, malaria is the commonest cause of death in children aged 0-4 years (Kofi, 1989). To curtail this problem, various malaria control efforts were initiated. These include: 1. The Ghana National Malaria Control Programme (NMCP), which works in collaboration with the Roll Back Malaria (RBM) programme and focused on the reduction of malaria burden by half by the year 2010. 2. The development of a model School-based Parasitic Diseases Control District (PDCD) in West Africa by the West African Centre for International Parasite Control (WACIPAC) (GPCI report, 2002). 3. The multidisciplinary case-control study of malaria pathogenesis and immunity in Ghana (Quakyi et al., 2004). It is interesting to note, however, that each of these projects had one important limitation, which was the reliance on microscopy as a standard diagnostic technique even though it is not rapid enough and may fail to detect some infections because of low sensitivity. There is therefore the need to develop new improved diagnostic techniques for better management of the disease. The Global Malaria Control Strategy (WHO, 1993) acknowledges that, prompt, rapid and accurate diagnosis is required to develop effective management strategies for prevention of severe and complicated disease. Although clinical diagnosis of malaria is relatively inexpensive to perform, fast, and requires no special equipment or supplies it may not be accurate enough (WHO, 2000). This is because, it relies on identification of clinical signs University of Ghana http://ugspace.ug.edu.gh 3 and symptoms which may also be caused by several other diseases, including, typhoid fever, influenza, meningitis, hepatitis, as well as haemorrhagic fevers (White, 1998; Bradley, 1996). Clinical diagnosis of malaria therefore, is based on a high level of presumption (WHO, 1991), following which the laboratory microscopic and/or other detective approaches are needed for confirmatory diagnosis. The conventional light microscopy is presently the established and most acceptable method for the laboratory confirmation of malaria (Katzin et al., 1991; Payne, 1988). Careful examination of a well-prepared and well-stained blood film by an expert microscopist is at the moment, the “gold standard” for specific detection and identification of malaria parasites (Payne, 1988). The microscopic method for diagnosis of malaria has several advantages (WHO, 2000). It is relatively inexpensive (Palmer et al., 1998), very specific, and the smears can provide a permanent record of the diagnostic findings and also be subject to quality control (Payne, 1988). However, several drawbacks such as inadequate sensitivity associated with microscopy make it unattractive enough for malaria diagnosis. There are already available rapid test kits {immunochromatographic tests (ICTs)} for alternative diagnosis of malaria. These techniques involve the detection of malaria parasite antigens in blood. They mostly employ the use of a dipstick or test strip, bearing monoclonal antibodies against malaria parasite antigens in immuno- chromatography. Immuno-chromatographic tests do not need any specialized expertise, and can be performed within 15 minutes. However, they are blood-based and therefore invasive. The development of a simple accurate rapid urine-based dipstick assay (RUBDA) for diagnosis of malaria could be very useful in overcoming these limitations. RUBDA is field- applicable and does not require any technical skill to perform. Lastly, the use of urine as test University of Ghana http://ugspace.ug.edu.gh 4 material makes the RUBDA a safer, more affordable, attractive and acceptable method for home-based management of malaria as compared to already available ones. Experience was drawn from earlier work in our laboratory, which led to the development of a field-applicable urine-based monoclonal antibody (MAb) dipstick for diagnosis of urinary schistosomiasis (Bosompem et al., 1997). The motivation for this study was drawn from reports that parasite antigens are excreted in the urine of patients of Chagas’ disease (Katzin et al., 1989); schistosomiasis (Bosompem et al., 1996a); and malaria (Oguonu et al., 2014; Katzin et al., 1988). Rodriguez- del Valle et al. (1991), also detected Plasmodium antigens and anti-malarial antibodies, in the urine of infected individuals and suggested that a urine-based assay for diagnosis of malaria would be feasible. In this study, malaria-related proteins were detected in the urine at concentrations high enough for direct analysis, and in view of this, it was suggested that a diagnostic assay based on the detection of specific antigens or antibodies in patients’ urine should be considered (Rodriguez-del Valle et al., 1991). 1.2 PROBLEM STATEMENT Obstacles to malaria control include delayed and inaccurate diagnosis and treatment; resistance of Plasmodium parasites and Anopheles vector mosquitoes to anti-malarial drugs and insecticides respectively as well as human behaviour that promote the proliferation of vector mosquitoes. Even though various strategies are being used to control malaria such as increased coverage of insecticide treated bed nets and intermittent preventive treatment for pregnant women, the morbidity and mortality attributable to malaria have not changed significantly. This situation emphasizes the need for improved tools to enhance the control efforts. University of Ghana http://ugspace.ug.edu.gh 5 For example, prompt, accurate, safe and affordable diagnosis is the first step towards effective management of malaria. However, the uncertain differential diagnosis associated with the routine use of clinical signs and symptoms of malaria necessitates the use of microscopy to confirm diagnosis even though the test is not sensitive enough. Furthermore, microscopy is labour-intensive, time consuming and requires well-trained technical staff for accurate diagnosis. Unfortunately, such highly trained technicians are usually not available at the peripheral levels of the health care delivery system (WHO, 2000) where most malaria- related deaths and morbidity occur. In view of this, Payne (1988) indicated that microscopic diagnosis of malaria using scarce resources could be doubtful. Existing immunochromtographic tests for malaria also have the weaknesses of invasive tests including exposure of health-workers to blood (Rodriguez-del Valle et al., 1991) and poor acceptance by some communities. Indeed, currently, none of the major tests for diagnosis of malaria can function without blood as a test material. This characteristic makes all these major tests unsuitable for home-based management of malaria (HBMM) in most of the low-income rural communities where most malaria-related deaths and morbidities occur. Perhaps, this is partly because, blood is used for pacification of gods and other rituals in these communities and therefore considered fetishism to engage in any practices that have connections with blood (Otabil, 1992). Also, with the connection of HIV/AIDS to blood, most people, would, under normal circumstances, not want to have anything to do with blood. The lack of effective diagnosis on account of these, therefore, makes the HBMM not to be effective enough. A simple dipstick test involving the use of urine as a test material would therefore be a welcome alternative. In addition, many pregnant women in rural, low-income communities do not report for antenatal healthcare (ANC), or report late, because of factors including inaccessibility and inability to meet service charges, thereby increasing their risk of malaria disease and death. University of Ghana http://ugspace.ug.edu.gh 6 However, in communities with inadequate healthcare infrastructure, simple field-applicable rapid diagnostic tools for malaria in pregnancy would lead to early detection and treatment of pregnancy malaria at the community and home-based level timely enough to avoid complications and/or death that could result. Therefore, a diagnostic technique, which combines high specificity, sensitivity, and rapidity to perform with non-invasiveness and cost- effectiveness, would be a most welcome alternative. It is in a bid to improve upon the limitations of the current diagnostic tools that this study proposed to produce an accurate rapid urine-based field-applicable dipstick test for diagnosis of malaria in infected individuals using Plasmodium antigens extracted from cultured parasites and urine of infected subjects. 1.3 OBJECTIVES 1.3.1 Main Objective The project aimed, at developing an accurate urine-based monoclonal antibody dipstick assay (RUBDA) for diagnosis of malaria utilizing Plasmodium parasite antigens from the urine of infected individuals. 1.3.2 Specific Objectives 1. Identification of malaria infected and uninfected subjects in an endemic population. 2. Extraction and characterization of biomarkers of malaria from infected human urine. 3. Generation and characterization of monoclonal antibodies against Plasmodium falciparum antigens using urinary and in vitro cultured parasites. 4. Reactivity of mouse anti-Plasmodium monoclonal and polyclonal antibodies in urine- based ELISA and western blotting assay. University of Ghana http://ugspace.ug.edu.gh 7 1.4 JUSTIFICATION The development of a simple, accurate, fast and non-invasive rapid urine-based dipstick assay (RUBDA) for diagnosis of malaria would lead to timely diagnosis and treatment of the disease, and so eliminate the complications and death that could result from delayed treatment of the disease. Finally, home-based management of malaria (HBMM) which is recognized as an important strategy for reducing the morbidity and mortality of the disease, thrives on accurate diagnosis outside the health facility, usually in the home by parents, caregivers and/ or by oneself. HBMM is, however, presently done using the presumptuous symptomatic diagnostic approach, because of lack of technical skills and logistics such as microscopes, which are required to perform the current blood-based tests. This situation, prevents the full utilization of the potential of the HBMM to combat the disease. The proposed urine-based test for malaria is simple, user friendly and lacks all the limitations mentioned above. Therefore, when developed and deployed commercially, application of the system would increase patronage of HBMM, and contribute greatly towards reduction of the alarming complications and death that are presently attributable to malaria. University of Ghana http://ugspace.ug.edu.gh 8 CHAPTER TWO LITERATURE REVIEW 2.1.0 MALARIA Malaria is a severe debilitating disease caused by obligate intracellular protozoan parasites of the genus Plasmodium. It exerts a heavy toll of illness and death (Ohalete et al., 2011; WHO fact sheet 2002), due to the immunological interactions with the human host as part of its life cycle. 2.1.1 Economic Cost of Malaria Economically, the cost of malaria is high. Even though the tropical regions of the world are the most affected, the impact of malaria on humans extends as far as the temperate zones (Nayyar et al., 2012). In areas where the disease is widespread, it is said to have major adverse economic effects on the inhabitants (Nayyar et al., 2012). Indeed, according to some authors, malaria is not just a disease commonly associated with poverty but also, it is a cause of poverty itselt and a major drawback to economic development (Worrall et al., 2005). Malaria was reported to be a major factor in the slow economic development of the American southern states in the late 19th and early 20th centuries (Humphreys, 2001). Also, between 1996 and 2002 for example, the total annual cost of treatment in Africa alone was estimated to be $US1.8 billion (WHO fact sheet 2002; Bradley, 1996). According to Nayyar et al. (2012), a comparison of average per capita gross domestic productivity (GDP) in 1995, adjusted for parity of purchasing power, between malaria endemic and non endemic countries gave a fivefold difference ($1,526 USD versus $8,268 USD). Also, in the period between 1965 and 1990, countries where malaria was common had University of Ghana http://ugspace.ug.edu.gh 9 an average per capita GDP that increased only 0.4% per year, compared to 2.4% per year in other countries without malaria (Sachs and Maloney, 2002). In Ghana, malaria mortality and morbidity have been reported to retard economic growth by reducing the capacity and efficiency of the labour force. In addition, a 10% reduction in malaria was associated with 0.3% growth in the economy; and for every increase in the morbidity rate of malaria, the GDP of Ghana is said to reduce by 0.41% (Asante and Asenso-Okyere, 2003). In monetary terms, this economic burden is said to transcend to US$1.92 million in the 2002 fiscal year alone. In its entirety, the economic burden of malaria has been estimated to cost Africa $12 billion (USD) per annum (Nayyar et al., 2012). This figure comprises the costs of health care, working days lost due to sickness, days lost in education, decreased productivity due to brain damage from cerebral malaria, as well as loss of investment and tourism (Greenwood et al., 2005). The degree of burden varies from country to country and is heavier in some countries, where it may be responsible for 30–50% of hospital admissions, up to 50% of outpatient visits, and up to 40% of public health spending (Roll Back Malaria WHO Partnership, 2003). 2.1.2 Geographic Distribution of Malaria and the Plasmodium Parasites Even though various reports have indicated different times of origin of malaria parasites, virtually all the writers believe that if not all, most of the present-day populations of the human malaria parasites originated from Africa (Conway et al., 2001). It is however important to note that irrespective of the origin of malaria and its causative Plasmodium parasites, the disease has generally, occurred in areas where environmental conditions allow parasite multiplication in the vector. Malaria today, therefore, is usually restricted to tropical and subtropical areas and altitudes below 1,500 m. In the past, University of Ghana http://ugspace.ug.edu.gh 10 however, malaria used to be endemic in much of North America, Europe and even parts of northern Asia, and today, it is still present on the Korean peninsula and in many other parts of Asia. This present pattern of geographic distribution of malaria could have been influenced by climatic changes and population movements. Plasmodium falciparum is the predominant species in the world. P. vivax and P. ovale are traditionally thought to occupy complementary niches, with P. ovale predominating in Sub-Saharan Africa and P. vivax in the other areas; but their geographical ranges do overlap. These two species are not always distinguishable on the basis of morphologic characteristics alone, and the use of molecular tools helps to clarify their diagnosis and exact distribution. P. malariae has wide global distribution, being found in South America, Asia, and Africa, but it is less frequent than P. falciparum in terms of association with cases of infection. P. knowlesi is found in south-east Asia. Climatic factors such as temperature, humidity and rainfall are the primary forces controlling the geographic distribution of malaria (Kelly-Hope et al., 2009; Data retrieved from http://www.cdc. gov/malaria/distribution_epi/distribution.htm on14th Jan 2010). This is because the survival and multiplication of the vector Anopheles mosquitoes, and the completion of the Plasmodium parasite life-cycle in the mosquito depend on favourable climatic conditions (Kelly-Hope et al., 2009). Malaria transmission therefore occurs in areas where climatic conditions are favourable. Environmental temperature is very important in the transmission and distribution of Plasmodium parasites. At temperatures below 20oC, for example, it is known that P. falciparum cannot complete its growth cycle in the vector Anopheles mosquito and therefore cannot be transmitted (Samuel et al., 2011). Also, it is generally known that in areas characterized by very high altitudes, cold seasons, hot, dry atmospheres, as found in some deserts (excluding the oases) where malaria parasites cannot survive and multiply, there is no malaria transmission. In warmer regions University of Ghana http://ugspace.ug.edu.gh 11 near the equator where humidity is high, malaria transmission is more intense and perennial (throughout the year), and P. falciparum predominates. Malaria transmission is therefore highest in tropical regions south of the Sahara, and high in subtropical regions in Africa, southern Asia, northern parts of South America, Central America as well as Indonesia, where the surrounding environmental factors are favourable. Figure 1 (Sachs and Malaney, 2002) and Table 1 (Zilversmit and Perkins. 2008) below illustrate the geographic distribution of malaria, the causative Plasmodium parasite and the vertebrate host. Figure 1: Global distribution of malaria (Sachs and Malaney, 2002) University of Ghana http://ugspace.ug.edu.gh 12 Table 1: Host and Geographic Distribution of some Plasmodium parasites Modified from Zilversmit and Perkins, 2008 Parasite Host Geographic Location P. falciparum, P. vivax, P. malariae Human Africa, Asia, South and Central America P. ovale Human Africa P. reichenowi Chimapanzee P. gonderi Madrill P. atheruri, P. vinkei, P. chabaudi, P. berghei, P. yoelii Rodent P. fieldi, P. simiovale, P. hylobati, P. inui, P. coatneyi, P. cynomolgi Macaque Southeast Asia P. knowlesi Macaque, Human P. gallinaceum Bird P. elongatum, P. relictum Bird Worldwide P. agamae, P. gigantum Lizard Africa P. floridense, P. azurophilum Lizard Carribbean/Central America P. mexicanum, P. chiricahuae Lizard North America P. faichildi Lizard Central America P. mackerassae Lizard Australia P. simium Spider Monkey South America P. brasilianum Spider/Howler/ Night Monkey Hepatocystis sp. Bat/Primate Africa, Asia Many temperate areas, such as Western Europe and the United States, used to be endemic for the disease, however, economic development and public health measures have led to the successful elimination of malaria even though most of these areas have Anopheles mosquitoes capable of transmitting the disease, and are therefore considered at risk of reintroduction of the disease (Kakkilaya, 2006). In all, the World Malaria Report 2005 stated that malaria is endemic in 107 countries throughout the world. Several countries in North Africa, the Eastern Mediterranean and Central Asia that have recently made tremendous progress in reducing transmission and are now within reach of eliminating malaria have been included in the malaria-endemic countries. University of Ghana http://ugspace.ug.edu.gh 13 However, countries that have only imported cases or occasional local transmission (introduced cases resulting from imported cases) are not included, although surveillance of malaria cases and provision of access to effective anti-malarial treatment remain important in these countries as well (WHO Malaria Report 2005). 2.1.3 The Biology and Life-Cycle of the Plasmodium Parasite 2.1.3.1 Structure of the Plasmodium The genome of Plasmodium falciparum, the most common causative agent for human malaria, has been sequenced completely, yielding 14 chromosomes and 5,300 genes. A large number of these genes enable the parasite to evade the host's immune defense mechanisms. The mapping of this genome sequence provides new avenues for research on possible vaccines. The sporozoite, the stage infective to humans, is 10-15 µm in length and about 1 µm in diameter. Each has a thin outer membrane, a double inner membrane below which lies the sub-pellicular microtubules. Figure 2 below illustrates the general morphology and ultrastructure of the P. falciparum parasite. The sporozoites, like the other motile invasive stages, the merozoites and ookinetes, are elongated and uni-nucleated. They all lack cilia and flagella except for the microgametes. However, they possess specialized secretory, locomotory and invasive organelles (rhoptries, micronemes and 3 polar rings) at their anterior end (Bannister and Sherman, 2009). These organelles constitute the apical complex required for penetration into host cells or tissues. The apical organelle complex gives these organisms the name Apicomplexa. The rhoptries are long, extending half the length of the body. The micronemes are convoluted elongated bodies which run into a common duct with the rhoptries at the anterior end of the body. University of Ghana http://ugspace.ug.edu.gh 14 Also, Plasmodium parasites possess one or more mitochondria, and an elongated membranous organelle known as the apicoplast (Waller and McFadden, 2005; Williamson et al., 2002) which is located near the posterior end of the body. Plasmodium parasites move by a unique form of gliding locomotion. Evolutionarily, they are closely related to ciliates and dinoflagellates (Hoppenrath and Saldarriaga (2012). Figure 2: General and Ultra-structure of the P. falciparum merozoite {from Bannister and Sherman (2009) and Bannister et al., (2003), respectively} 2.1.3.2 Life Cycle of the Plasmodium Parasite The Plasmodium genus of protozoan parasites has a life cycle, which is split between a vertebrate host and an insect vector (Bradley, 1996). The female Anopheles mosquito is the vector for transmission. Mosquitoes in general, feed on nectar or any edible sugar-containing fluid or solution found in the environment. The female species, however, require a protein of the vertebrate blood for development of their ovaries and maturation of the fertilized eggs, hence the need to feed on vertebrate blood (Brown et al., 2008). The schematic representation of the life cycle of the parasite is shown in figure 4 below. The life cycle begins with the injection of spindle shaped infective sporozoites from the vector mosquito salivary gland into the human body (tissue or blood capillary) with mosquito anticoagulant saliva which serves to prevent clotting of blood in the mosquito proboscis and ensures an even flowing meal. It has been estimated by various methods University of Ghana http://ugspace.ug.edu.gh 15 (Ponnudurai et al., 1991) that compared to the total number of sporozoites observed in the salivary gland, only about 10% (<50) sporozoites are injected into the vertebrate host during each blood meal. Figure 3: Life cycle of the Plasmodium falciparum malaria parasite in the human and the mosquito hosts (Bannister and Sherman, 2009) The traditional perception has been that once the sporozoites have been injected into the peripheral circulation of the human host, they are carried into the liver, where they penetrate liver cells (hepatocytes), remain for 9-16 days, become ring shaped (schizonts) and multiply within the cells (Doolan et al., 2009). However, other studies on the kinetics of sporozoite migration (Yamauchi et al., 2007), have shown that sporozoites are first injected by mosquitoes into the skin, where they remain for up to 6 hours before trickling into the bloodstream, before migrating into the liver. Also, Amino et al. (2006), reported that approximately one-third of the sporozoites leaving the site of inoculation may enter lymphatic University of Ghana http://ugspace.ug.edu.gh 16 vessels and finally end up in the regional lymph nodes. The findings of these studies therefore suggest that there are multiple potential sites for sporozoite-host interaction (Doolan et al., 2009). In the liver, the sporozoites penetrate liver cells, become transformed into schizonts that multiply asexually. P. falciparum and P. malariae sporozoites trigger immediate schizogony whereas P. ovale and P. vivax sporozoites may either trigger immediate schizogony or have a delayed trigger, resulting in dormant hypnozoites, also referred to as merosomes (Bradley, 1996; Sturm et al., 2006; Liu and Tang, 2012). Some of the schizonts return into the blood stream in a form called merozoites and invade red blood cells (RBCs). Within the RBCs, they transform into feeding stage plasmodia called trophozoites that feed on the haemoglobin and multiply to produce more merozoites which re-infect the liver. It may take about 48 hours for P. falciparum to go through this erythrocytic schizogony. On the other hand, the trophozoites may transform again into sexual stages called micro- (male) and macro- (female) gametocytes, which have no further activity within the human host. The P. falciparum parasite takes about 10-12 days to complete gametocytosis. The gametocytes may then be sucked up into the gut of a mosquito during another blood meal. Here, ex-flagellation of micro-gametocytes and transformation of macro-gametocytes occur, giving rise to micro (male) gametes and macro (female) gametes respectively. Subsequently, fertilization of the macro-gamete by the micro-gamete occurs, leading to the formation of a zygote. The resulting zygote then develops into a motile ookinete, which penetrates the cell wall of the mosquito mid-gut lining, and develops into an oocyst. Sporogony within the oocyst inside the mosquito is governed by environmental temperature, as Anopheline mosquitoes are poikilotherms (Samuel et al., 2011). It produces many sporozoites, which are liberated when the oocyst ruptures. After rupturing out of the cyst, the sporozoites escape into University of Ghana http://ugspace.ug.edu.gh 17 the haemocoele and migrate to penetrate salivary gland cells, where they lie in vacuoles for up to 59 days. Here, they mature to become up to 1000 times more infective and more antigenic, with the circumsporozoite antigen (CSA) being the predominant antigen. They then remain in this form until they are injected into the human, or another vertebrate host, during another blood meal to repeat the cycle. 2.1.4 Clinical Signs and Symptoms of Malaria Generally, symptoms of uncomplicated malaria are flu-like (Bartoloni and Zammarchi, 2012) and include headache, fever and generalized body pains, chills, perspiration, anorexia, vomiting, malaise, arthralgia (joint pains) and sometimes, abdominal pains, diarrhoea and (White, 1998; Bartoloni and Zammarchi, 2012). Clinically, the spleen and liver are often palpable, and may be misdiagnosed as influenza (Bartoloni and Zammarchi, 2012), and other conditions such as septicemia, gastroenteritis and viral diseases (Nadjm and Behrens, 2012) in non-endemic areas, and, unless treated promptly, the clinical picture can deteriorate rapidly (WHO, 1991). Severe and complicated malaria results, primarily, from delay in treatment of an uncomplicated P. falciparum attack (Bradley, 1996) although the most virulent of human malaria parasites are P. falciparum and P. vivax (White, 2003). Plasmodium parasites infect a variety of vertebrate hosts including primates, rodents, ungulates, birds, and lizards, however, they rarely cause severe disease in any vertebrate hosts apart from humans (Zilversmit and Perkins. 2008). A patient with severe and complicated malaria will often present with impaired consciousness, weakness and jaundice (Bartoloni and Zammarchi, 2012). Other complications include cerebral malaria (Bartoloni and Zammarchi, 2012; Brewster et al., 1990), generalized convulsions, normocytic anaemia, hemolytic anemia, retinal damage renal failure, hypoglycaemia, fluid-electrolyte and acid-base disturbances, pulmonary oedema, circulatory University of Ghana http://ugspace.ug.edu.gh 18 collapse, shock, disseminated intravascular coagulation, hyperpyrexia, hyperparasitaemia, malaria haemoglobinurea and death (WHO, 1991; Bartoloni and Zammarchi, 2012). 2.1.5 Pathogenesis of Malaria Under certain circumstances Plasmodium infection causes severe disease such as anemia or cerebral malaria (Horata et al., 2009). The expression of disease is influenced by both parasite and host factors (Van der Heyde et al., 2006), as exemplified by the worsening severity of disease during pregnancy. Pathogenesis relates to the various host (host immunological processes) and parasite factors that are responsible for injury/ damage to host cells/ tissues. The pathogenesis of severe malaria therefore involves a cascading interaction between parasite and host red cell membrane products, cytokines and endothelial receptors, leading to inflammation, activation of platelets, hemostasis, a procoagulant state, microcirculatory dysfunction and tissue hypoxia, resulting in various organ dysfunctions manifesting in severe malaria (Van der Heyde et al., 2006). Understanding the pathogenesis of malaria is essential because, it enhances our ability to develop strategies to prevent the most severe forms of malaria. The liver stage of Plasmodium infection does not cause any disease, though liver cells are infected. The pathogenic process occurs only during the erythrocytic cycle. During this stage, there is a huge, periodic increase in parasite populations that may enhance the probability of differentiation to gametocytes, the stage infectious to mosquitoes. A peculiarity of P. falciparum is its ability to cause erythrocytes infected with maturing parasites to adhere to endothelium of venules (cyto-adherence). Central to the interaction between the host’s immune system and the parasite factors that leads to pathogenesis are cytokines which are released by immunocompetent cells in a highly regulated fashion (Clark et al., 2006; Van der Heyde et al., 2006). University of Ghana http://ugspace.ug.edu.gh 19 The the pathological symptoms of malaria begin with the invasion of the erythrocytes by the merozoites and the rupture of erythrocytes releasing Plasmodium parasite soluble products (malarial toxins) which trigger systemic release of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and Interleukins (IL1α, 1β and IL6) into the host blood stream (Clark et al., 2006; Van der Heyde et al., 2006). The presence of malaria toxins and antigens, and the release of TNF-α, IL platelets in the body cause inflammation marked by increase in body temperature above 37.5oC (fever) and pain in the host. The pro-inflammatory cytokines are also involved in cerebral malaria (Van der Heyde et al., 2006). During cerebral malaria, infected RBCs adhere to the inner wall of the blood vessels and block the blood capillaries of the brain (Van der Heyde, et al., 2006). Occlusion of the capillaries interrupt the flow of nutrient and oxygen, and also disrupts brain function that may lead to coma and death (Idro et al., 2010). Also, owing to the cleaning behavior of the spleen which filter off infected RBCs, ruptured RBC membranes and malaria pigment from the blood, it may swell up (splenomegaly) and become brownish in colour. The architecture of the white pulp becomes markedly disorganized, with dissolution of the marginal zone and relative loss of B cells (Urban et al., 1999). Though rare, malarial splenomegaly may also lead to pathological splenic rupture (Imbert et al., 2009; Cinquetti et al., 2010). Anemia is caused by the destruction of erythrocytes (RBCs) during asexual replication of the parasites. This process leads to tremendous multiplacation of parasites with densities that may reach 400,000 parasites/µl of blood per patient (Halbert et al., 2010). As these parasites multiply and feed on the RBCs, they degrade and rapture resulting to reduction in RBC concentration in the blood, a condition which is called anemia. Anemia may also be caused by the absorption of antigen on non-parasitized erythrocytes with complement- University of Ghana http://ugspace.ug.edu.gh 20 mediated lysis and suppression of hematopoiesis by cytokines. In some cases, the therapy- associated hemolysis induced by medications in G6PD deficiency may also lead to anemia. Hypoglycemia is common in malaria. Malaria parasitized erythrocytes utilize glucose 75 times faster than uninfected cells although there is no direct correlation between hypoglycemia and parasitaemia. In addition, treatment with quinine and quinidine stimulate insulin secretion, thereby reducing blood glucose in the brain. Parasites sequester (cytoadhere) themselves also in the placenta, the lung, the liver, the kidney and subcutaneous tissues (Van der Heyde, et al., 2006). The cyto-adherence to many endothelial receptors in such tissues is mediated by variants of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, which are anchored at the red cell membrane skeleton by the knob-associated histidine-rich protein and expressed at the surface of the infected erythrocytes (Maier et al., 2008; Miller et al., 2002). The presence of this protein on the surface of infected RBCs creates surface changes that allow parasite encoded adhesion and sequestration into host cells (Smith et al., 2001) PfEMP1 is the product of the var gene family. It undergoes antigenic variation (diversity) and thus plays a major role in the pathogenesis of malaria (Horata et al., 2009). The extracellular region of PfEMP1 possesses multiple adhesion domains which promotes binding to a broad range of endothelial cell receptors, including CSA, ICAM1, and CD36 for the placenta, the brain and the vascular endothelium. The severity of malaria is observed mostly in pregnant women and children below 5 years of age (WHO Malaria Policy Meeting Report, 2012; Hviid, 2007) because of lack of adequate immune protection against the disease (Hviid, 2007). In areas where P. falciparum is endemic, women who normally have immune protections against malaria tend to lose this protection during pregnancy (Hviid, 2007). The supposed reduction in immunity during pregnancy is attributed to a transient depression of cell-mediated immunity to allow retention University of Ghana http://ugspace.ug.edu.gh 21 of foetal allograft, which unfortunately, also causes a reduction in resistance to various infectious diseases, including malaria (Meeusen et al., 2001). This condition causes both the frequency and the severity of the disease to be higher in pregnant than non-pregnant women (WHO, 1983). During pregnancy, especially, in primigravidae, P. falciparum infected RBCs coated with pfEMP1 of CSA-binding capacity, become attracted to and sequestered in CSA within the placenta. The aggregation of P. falciparum infected erythrocytes bearing pfEMP1 and uninfected erythrocytes in tissues occlude fine blood capillaries and ultimately blocks blood flow, limits the local oxygen and nutrient supply. The aggregation of P. falciparum infected erythrocytes bearing pfEMP1 and uninfected erythrocytes in tissues occlude fine blood capillaries and ultimately blocks blood flow, limits the local oxygen and nutrient supply, hampers mitochondrial ATP synthesis, and stimulates cytokine production thereby causing abnormal functioning of host cells and tissues to which the pfEMP1 are attached. All of these factors contribute to the development of severe disease, hence the obstetric complications and death of pregnant women (Clark et al., 2006; Horata et al., 2009; Anstey et al., 2009), or intra-uterine growth retardation (Poovassery et al., 2009), leading to pregnancy failure, miscarriages or delivery of low birth-weight babies (Poovassery et al., 2009), which is an important risk factor for infant mortality (Steketee et al., 2001). 2.1.6 Classification of the Plasmodium Parasite According to Bannister and Sherman (2009), Plasmodium is currently classified on the basis of morphological and molecular evidence as: Kingdom Protozoa, Subkingdom Biciliata, Infra kingdom Alveolata, Phylum Myzozoa, sub-phylum Apicomplexa. The Subphylum Apicomplexa, which are so named because of the possession of the apical University of Ghana http://ugspace.ug.edu.gh 22 organelle complex at the anterior end of the parasite for locomotion and penetrating into host cells and tissues. It comprises nearly 5000 described species, all parasitic including several genera of medically and economically important organisms such as Plasmodium, Babesia, Toxoplasma, Cryptosporidium, Theileria, Eimeria and Isospora. Three Classes of organisms fall under Apicomplexa (Escalante and Ayala, 1995), with Plasmodium belonging to the class Aconoidasida, Order Haemosporina, and Genus Plasmodium. The genus Plasmodium was first described by Ettore Marchiafava and Angelo Celli in 1885. Currently, with new species being continuously identified and described, it is said to comprise over 200 species (Chavatte et al., 2007) which are all parasites of higher order organisms. With increasing understanding of the evolution and systematics of malaria parasites over the past 20 years, Martinsen et al., 2008, indicated that the genus Plasmodium may not to be monophyletic, and includes parasites of other genera including Hepatocystis. The parasite is known to have two hosts in its life cycle which comprise a vector, usually a mosquito and a vertebrate host, ranging from humans to monkeys, rodents, bats, birds, and reptiles (Martinsen et al., 2008; Vargas-Serrato et al., 2003). According to Bradley (1996), with the exception of P. malariae, which may also infect the higher orders of primates, four distinct species of Plasmodium parasites were originally known to infect humans, and included, P. falciparum, P. vivax, P. malariae and P. ovale. However, Kakkilaya (2006) reported that by 1966, 10 species of Plasmodium, naturally present in monkeys and apes had been shown to be capable of infecting humans. In corroboration of Kakkilaya (2006)’s report, molecular studies recently revealed the possible existence of additional parasite species or morphological variants of existing species that also infect humans (Sutherland et al., 2010). This caused the number of Plasmodium parasites now known to infect humans to be at least eleven (Sutherland et al., 2010). For example, sequencing of the gene for the Plasmodium circumsporozoite surface protein (CSP) showed University of Ghana http://ugspace.ug.edu.gh 23 that some individuals initially thought to have been diagnosed with P. vivax infections were actually infected with a distinct species more closely related to P. simiovale, a simian malaria parasite which is morphologically identical to P. vivax. Consequently, this species was named the P. vivax-like. In addition, molecular analysis has indicated that P. ovale consists of two clades that are as divergent as distinct species and has suggested that they should be designated as sub- species P.o. curtisi and P.o. wallikeri (Oguike et al., 2011). Similarly, molecular analyses have indicated that some morphological variants of P. malariae that infect humans are distinct parasites related to P. malariae and P. brasilianum. P. brasilianum is a simian parasite of the South and Central America which is often speculated to have originated from humans as a result of colonization of the New World. Furthermore, molecular studies showed that nearly all the humans diagnosed to be naturally infected with P. malariae in Malaysia, were actually harbouring the simian malaria parasite, P. knowlesi (Cox-Singh and Singh, 2008). Also, four fatalities associated with P. knowlesi infection were reported in Malaysia, and this finding coupled with other naturally occurring human infections of P. knowlesi elsewhere therefore indicate that human infections with P. knowlesi may not be a rare occurrence but may be widespread in Malaysia and perhaps other parts of southeast Asia (Cox-Singh and Singh, 2008). It is interesting to note however that while some of these current taxonomic names are repetitions or confirmation of earlier nomenclature, the molecular evidence has introduced new dimensions of taxonomic names which were not part of the above. This observation, in accordance with scientific discovery, points to the fact that as additional elucidation of the characteristics of organisms is obtained from further studies, the taxonomy of Plasmodium, like that of all other organisms will continue to change. University of Ghana http://ugspace.ug.edu.gh 24 2.2.0 ANTIBODIES Antibodies are large Y-shaped glycoprotein molecules produced and secreted by specialized white blood cells or plasma cells known as B lymphocytes (B cells) which form part of the humoral arm of the host immune system (Pier et al., 2004; Lipman et al., 2005). They are very essential in immunity. They are one of the principal effectors of the adaptive immune system and are deployed by the immune system in response to the invasion of the host body by foreign objects such as bacteria and viruses (Lipman et al., 2005). They are then used by the immune system to identify and destroy such foreign matter, thereby preventing them from causing disease or injury to the body (Lipman et al., 2005). Each of the bi-forked ends of the Y-shaped antibody has a structural receptor like a lock known as a paratope or antigen binding site (Figure 4a and b below) to which a specific epitope of an antigen is attached before destruction. By the ‘lock and key’ attachment or binding mechanism, antibodies mark infectious agents, microbes or infected body cells before they are neutralized or destroyed by other parts of the immune system. Antibodies may occur in two physical forms, one being a soluble form secreted by B-cells and found in the body circulation, and the other being membrane bound and attached on the surface of B-cells and is therefore known as the B-cell receptor (BCR). 2.2.1 The Basic Structure of Antibodies Antibody molecules are heavy globular plasma glycoproteins comprising one or more basic structural units called immunoglobulin (Ig for short) monomers. Each Ig monomer is a "Y"-shaped molecule that consists of four polypeptide chains; two identical heavy (H) chains and two identical light (L) chains joined together by disulfide and non-covalent bonds (Lipman et al., 2005). Each chain comprises structural domains called immunoglobulin University of Ghana http://ugspace.ug.edu.gh 25 domains containing about 70-110 amino acids, some of which are attached to sugar chains thereby giving the antibody its glycoprotein properties. Some of the structural or Ig domains at the amino acid end, show considerable variation in amino acid composition and are referred to as the variable (V) regions to distinguish them from the relatively constant (C) carboxylic acid regions. One light chain has an approximate molecular weight of about 50 kDa. Each heavy chain on the other hand, has about twice the number of amino acids and molecular weight (~50-55,000) as each light chain (Janeway et al., 2001; Lipman et al., 2005). This gives the immunoglobulin monomer a molecular weight of approximately 150 kDa (150,000). The amino acid sequence in the tips of the "Y" varies greatly among different antibodies. The variable region includes the ends of the light and heavy chains. Each variable region consists of 110-130 amino acids (Janeway et al., 2001; Mayer, 2009) or 220 amino acids (110 for each of the heavy and light chains) (Lipman et al., 2005). It gives the antibody its specificity for binding antigen. The tip of each arm of the Y, for example, has receptors that can bind to two identical antigens and, therefore, are specific to such antigens. This region of the antibody is called the antigen binding fragment (Fab region). It consists of one constant and one variable domain from each heavy and light chain pair of the immunoglobulin molecule, which makes each antibody molecule at least bivalent. The structure of the paratope at the amino terminal is determined by the mode of arrangement of amino acids in the variable domains of each heavy and light chain pair. The antibody uses each of these antigen binding sites or paratopes to bind to an epitope on an antigen. The base of the Y of the immunoglobulin molecule is called the crystallizable fragment (Fc region). It is composed of two heavy chains that may provide two or three constant domains depending on the class of the antibody (Janeway et al., 2001; Mayer, 2009). It plays a role in modulating immune cell activity by ensuring that each antibody generates an University of Ghana http://ugspace.ug.edu.gh 26 appropriate immune response for a given antigen. It achieves this by binding to a specific class of Fc receptors, and other immune molecules, such as complement proteins. Through this process, different biological effector functions are mediated. They include activation of natural killer cells and classical complement pathway, lysis of cells, phagocytosis, recognition of opsonized particles, and degranulation of mast cells, basophils and eosinophils. These functions therefore determines the mechanisms used in destroying the antigens. 2.2.1.1 Heavy chain immunoglobulin There are five known types of mammalian Ig heavy chains or isotypes, which are denoted by the Greek letters: α, δ, ε, γ and μ (Janeway et al., 2001). The type of heavy chain present defines the class of the antibody (Mayer, 2009). Consequently, the 5 heavy chain types lettered α, δ, ε, γ and μ have been given immunoglobulin class names that combine “Ig” with the initials of the names of the corresponding Greek letters as IgA, IgD, IgE, IgG, and IgM respectively. Distinct heavy chains differ in size and composition such that α and γ for example contain approximately 450 amino acids, while μ and ε have approximately 550 amino acids (Shimizu, 2004; Mayer, 2009). Unlike mammals, birds, have only one known major serum antibody which is also found in the egg yolk and called IgY. Even though it has a partial resemblance to the mammalian IgG it is quite different from it. This partial resemblance caused the bird IgY to be called IgG in some older literature and even on some commercial life sciences product websites "IgG", which however, is incorrect and can be confusing. Each heavy chain has two regions, the constant region and the variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains γ, α and δ have a constant region composed of 3 Ig domains in succession (in tandem), and a hinge region that confers additional. Heavy chains μ and ε, University of Ghana http://ugspace.ug.edu.gh 27 on the other hand, have a constant region composed of 4 immunoglobulin domains (Janeway et al., 2001). The variable region of the heavy chain differs in antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or B cell clone and is composed of a single Ig domain. 2.2.1.2 Light chain immunoglobulin Two types of immunoglobulin light chain occur in mammals which are the lambda (λ) and kappa (κ) (Mayer, 2009). Each light chain has two domains in series, one being constant and the other one, variable. Each ligh chain is approximately 211 to 217 amino acids long (Janeway et al., 2001; Shimizu, 2004; Mayer, 2009). As mentioned above, each mammalian immunoglobulin monomer contains two light chains that are always identical, and only one type of light chain, κ or λ, is present per antibody. Other types of light chains, such as the iota (ι) chain, have been reported in other vertebrates such as bony fishes (Teleostei) and sharks (Chondrichthyes). The most significant region for antigen binding is the variable domain which is also referred to as the FV region. Here, the actual receptors responsible for binding to antigen epitopes are 3 hyper-variable loops of β-strands, each comprising 5-10 amino acids in length also known as the complementarity determining regions (CDRs), which are located on each of the heavy (VH) and light (VL) chains (Lipman et al., 2005). University of Ghana http://ugspace.ug.edu.gh 28 Figure 4a Figure 4b Figure 4a and b: Basic structures of the immunoglobulin molecule (retrieved from “http://en.wikipedia.org_/wiki/File:Immunoglobulin_basic_unit.svg” and “http://www.biology.arizona.edu/immunology/tutorials/antibody/structure.html” respectively, on 2-2-2013. 1 is the Fab region of the antibody; 2 is the Fc region; 3 is the Heavy chain (long, bow-jointed units) with one variable (VH) domain followed by a constant domain (CH1), a hinge region, and two more constant (CH2 and CH3) domains; 4 is the Light chain (short unit) with one variable (VL) and one constant (CL) domain; 5 is the Antigen binding site (paratope) and 6 is the Hinge regions. 2.2.2 Classification of Antibodies As stated above, 5 different primary antibody classes are known in mammals, which are IgG, IgM, IgA, IgD and IgE. They perform different roles, and help direct the appropriate immune response for each different type of foreign object encountered (Lipman et al., 2005). In avians, however, there are three classes which are IgY, IgM, and IgA (Lipman et al., 2005), which are distinguished by the type of heavy chain they possess. IgG molecules have heavy chains known as gamma-chains; IgMs have mu-chains; IgAs have alpha-chains; IgEs have epsilon-chains; and IgDs have delta-chains. Differences in the constant region structure marked by differences in the amino acid sequence of the heavy chain polypeptides allow each immunoglobulin to function in a different type of immune response and at a particular stage of the immune response. The University of Ghana http://ugspace.ug.edu.gh 29 polypeptide protein sequences responsible for these differences are found primarily in the Fc fragment. Each of the five different mammalian primary antibody classes or heavy chains, in a selected group of mammals, IgG and IgA are further subdivided into subclasses, referred to as isotypes, due to polymorphisms in the conserved regions of the heavy chain (Lipman et al., 2005). Unlike the heavy chains, there are only two main types of light chains: kappa (κ) and lambda (λ). Antibody classes differ in valency as a result of different numbers of Y-like units (monomers) that join to form the complete protein. For example, in humans, functioning IgM antibodies have five Y-shaped units (pentamer) containing a total of ten light chains, ten heavy chains and ten antigen-binding sites (Table 2, below). The Ig class determines both the type and the temporal nature of the immune response (Lipman et al., 2005). University of Ghana http://ugspace.ug.edu.gh 30 Table 2: Classification of antibodies based upon location, structure and function Immunoglobulin Class Types Location and/ or Function Antibody Complexes Structure IgA 2 Found in mucosal areas, such as the gut, respiratory tract and urogenital tract, and prevents colonization by pathogens (Schiff et al., 1986). Also found in saliva, tears, and breast milk. Dimer (dimeric) IgD 1 Functions mainly as an antigen receptor on B cells that have not been exposed to antigens. It has been shown to activate basophils and mast cells to produce antimicrobial factors. Monomer (monomeric) IgE 1 Binds to allergens and triggers histamine release from mast cells and basophils, and is involved in allergy. Also protects against parasitic worms (Pier et al., 2004) Monomer (monomeric) IgG 4 In its four forms, provides the majority of antibody-based immunity against invading pathogens (Pier et al., 2004). The only antibody capable of crossing the placenta to give passive immunity to the fetus. Monomer (monomeric) IgM 1 Expressed on the surface of B cells (monomer) and in a secreted form (pentamer) with very high avidity. Eliminates pathogens in the early stages of B cell mediated (humoral) immunity before there is sufficient IgG (Pier et al., 2004). Pentamer (Pentameric) University of Ghana http://ugspace.ug.edu.gh 31 2.2.3 Classification of Antibodies According to Multiplicity of Clones Apart from the type of antibody classification presented above, antibodies may also be categorized into polyclonals or monoclonals based on the multiplicity of epitopes recognized, and regarding this mode of classification, the characteristics of antibodies are presented as follows. 2.2.3.1 Polyclonal Antibodies Polyclonal antibodies are a collection of heterogeneous antibodies originating from multiple B cell clones of an immunocompetent animal that has been immunized with specific antigens (Nakazawa et al., 2010). As a result of their origin from B cell clones of different genetic conformation, they are the outcomes of various biochemical mechanisms of combination and recombination of heavy and light chains that give rise to different binding site characteristics with the ability to bind to a diverse spectrum of antigens, or multiple epitopes on the same antigen (Lipman et al., 2005; Nakazawa et al., 2010). Like MAbs, they are produced by inoculating an animal such as a goat, sheep, or rabbit with a specific antigen that elicits a primary immune response. Following this, a secondary and tertiary immunization produces higher titres of antibody against the particular immunogen. However, unlike the case for MAb production (Pandey, 2010; Yokoyama, 1999), the serum of the animal containing the polyclonal antibodies is harvested (Zhang et al., 2010; Sevier et al., 1981) and purified by affinity chromatography in order to enrich the antibodies generated (Zhang et al., 2010; Josic and Lim, 2001). This process ultimately leads to the production of high titre and high affinity polyclonal antibodies against the antigen of interest. A larger animal such as a goat or a sheep is preferred for polyclonal antibody production because the yield of antibodies per animal is high (Leenaars and Hendriksen, 2005). University of Ghana http://ugspace.ug.edu.gh 32 Unlike monoclonal antibodies, polyclonal antibodies cannot be collected for an indefinite amount of time and often require multiple animals to be immunized with the same antigen. Antibodies are harvested from each animal; however, animals immunized with the same antigen develop differential immune responses and this could result in variability in polyclonal antibody production between batch preparations. Since each antibody preparation is collected from multiple B cell clones of different genetic characteristics, polyclonal antibodies are capable of recognizing different epitopes within the antigen and bind the antigen with varying affinities (Nakazawa et al., 2010). This can prove to be advantageous in many biological assays depending on the particular application. For example, polyclonal antibodies may cross-react with antigens that share high homology, which can be suitable when trying to detect known or unknown isoforms of an antigen (Lopata and Cleveland, 1987). In addition, they can be utilized to enhance the detection level of a particular antigen since multiple antibodies will bind the same antigen at epitope specific regions. This in turn can be valuable when attempting to detect low expressing antigens. 2.2.3.2 Monoclonal Antibodies Monoclonal antibodies (MAbs) are a population of antibodies that recognize a single epitope of an antigen. They are produced from a single B lymphocyte (cell) clone of an immunized animal, thereby generating a clonal population of antibodies, identical to one another and all recognizing the same epitope of a specific antigen. MAbs were first detected in sera of patients with multiple myeloma in which clonal expansion of malignant plasma cells produce high levels of an identical antibody resulting in a monoclonal gammopathy (Lipman et al., 2005). The procedure for MAb production is in many ways similar to the isolation of a cDNA from a cDNA library. University of Ghana http://ugspace.ug.edu.gh 33 However, in the case of MAbs, an immunized animal produces a library of B cells that are secreting antibodies of different specificities. The B cells can be isolated from the spleen of the animal and fused with myeloma cells to produce multi-specific antibody producing hybridoma cells. To obtain the MAbs therefore, a single B lymphocyte-myeloma hybrid cell (hybridoma cell) that is producing the antibody of required specificity is carefully selected and multiplied in vitro into a population of cells that can secrete a large amount of the desired antibody. In practice, an animal such as a mouse or a rabbit is injected with a specific antigen to induce a primary immune response (Leenaars and Hendriksen, 2005), followed by a secondary and tertiary immunization to produce antigen-specific B cells that generate and secrete higher titres of antibody against the inoculated antigen (Leenaars and Hendriksen, 2005). Usually, the animal of choice is the BALB/c mouse because many of the myeloma cells available for cell fusion are of the BALB/c origin (Leenaars and Hendriksen, 2005). The spleen of the immunized laboratory animal is removed aseptically and minced to free the splenocytes (B lymphocytes). Although antibody producing B cells alone can produce antibodies, characteristically, they have a short lifespan and cannot be easily cloned and expanded in vitro which eventually leads to their cessation of antibody production. Also, even though myeloma cells have the capacity to grow continuously in vitro they cannot produce antibodies. Therefore, by fusing specific antibody-producing B cells and myeloma cells together, a hybrid of the two (hybridoma) cells results which possesses both the ability to grow perpetually in vitro and also produce unique monoclonal antibodies, thereby causing the limited lifespan of the B cells to be prolonged indefinitely. The immortalized B lymphocyte-myeloma-hybrid cell (hybridoma cell) that results then provides a constant supply of highly specific monoclonal antibodies. Since monoclonal University of Ghana http://ugspace.ug.edu.gh 34 antibodies only recognize one epitope (Deb et al., 2013), they generally have low cross reactivity with non-specific antigens. Their epitope specificity, limited cross reactivity, and long term yield make monoclonal antibodies attractive for use in many biological assays and applications. This strategy for generating monoclonal antibodies of a desired specificity was invented by the two researchers, Georges J. F. Köhler and Cesar Milstein in the mid-1970s for which they were awarded the Nobel prize (Köhler and Milstein, 1975; Lipman et al., 2005; Deb et al., 2013). This technique was the first practical method for mass-production of monoclonal antibodies in vitro by inducing cells of the immune system to produce pure antibodies against a chosen antigen. Their breakthrough is considered one of the most important techniques of biotechnology as it led to the advancement of the use of antibodies to achieve various objectives. 2.2.3.2.1 The Hybridoma Principle and Mechanism of Reaction Splenocytes are B-lymphocytes. They secrete antibodies but cannot survive in vitro. Myeloma cells are tumour-like or cancerous cells that have been conditioned to possess the following characteristics. First, they have been treated with 8-Azaguanine which aborts the Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene and inhibits HGPRT enzyme activity in the cells. HGPRT catalyzes the alternative/ salvage reaction pathway for cell metabolism and nucleotide synthesis. Its inhibition or absence therefore makes the cells susceptible to destruction by aminopterin which is a folic acid antagonist. Also, myeloma cells have been immortalized to make them survive in vitro perpetually. In addition, they are carefully selected to ensure that they do not secrete antibodies themselves (Leenaars and Hendriksen, 2005). Furthermore, they are treated with a marker that enables non fused parental cells to be University of Ghana http://ugspace.ug.edu.gh 35 removed after the fusion is completed. Fusing B cells and myeloma cells together results in a hybrid cell, the hybridoma cell which can secret antibodies and survive in vitro. The fusion solution contains HAT-selection medium (Pandey, 2010) which is prepared by mixing Hypoxanthine and Thymidine (precursors for DNA synthesis) with a normal culture medium, and later adding Aminopterin which is a protease inhibitor. Myeloma cells and B lymphocytes from the spleen of an immunized mouse are mixed together according to laid down procedures. Polyethylene glycol (PEG) is then added to concentrate and fuse the cells together (Pandey, 2010). Following this, HAT selection medium is added. The Aminopterin in the HAT medium blocks the synthesis of nucleotide precursors in both B-lymphocytes and myeloma cells, which adversely affect the cells, making them unable to make any purine nucleotides. The cells in this condition, are thus forced to use the salvage (alternative) pathway for protein synthesis (Pandey, 2010). Also, the abortion of HGPRT activity in myeloma cells due to treatment with 8- Azaguanine prevents them from undergoing the alternative pathway and hence cell division leading to their death. Thus, addition of PEG causes the cell membranes of the B-lymphocytes and myeloma cells to fuse allowing the cytoplasmic contents to mix but not the nuclei. After cell fusion, cellular products containing only myeloma nuclei will abort because they cannot metabolize either by the forward or salvage reaction pathway for protein synthesis. Also, cells containing B-lymphocyte nuclei only, will abort because, by their nature, B cells cannot survive in vitro for long (Pandey, 2010). Finally, only daughter (hybridoma) cells with both the myeloma and B cell characteristics can survive and multiply in vitro because, they have the myeloma cell nature of immortality as well as the B cell characteristics of being able to undergo the alternative pathway for protein University of Ghana http://ugspace.ug.edu.gh 36 synthesis as a result of the presence of a functional HGPRT gene, and multiply in vitro (Pandey, 2010). 2.2.3.2.2 Hybridoma Cell Cloning by Limiting Dilutions Method The next, and most experimentally challenging, line of activity is to design a method of selecting, isolating and multiplying (cloning) single (monoclonal) cells that produce antibodies of interest (Pandey, 2010). Usually, this is done by the method of limiting dilution (Pandey, 2010). In practice, the culture is divided into many individual smaller cultures, each containing a single cell which divides into a colony of several hundreds of cells. The supernatant from each colony of culture is then sampled and assayed for the antibody of interest. The methods used are immunoassay techniques that allow antibodies which are monospecific and have very high sensitivity to the antigen of interest to be selected (Dewar et al., 2005; Lipman et al., 2005). The selected monoclonal hybridoma cell with qualities of interest is then up-scaled, multiplied or expanded (Leenaars and Hendriksen, 2005) to produce several cultures of hybridoma that generate large quantities of the desired antibodies. 2.2.3.2.3 Monoclonal Antibody production by Ascites Production Method After a single hybridoma cell that secretes monoclonal antibodies of desired sensitivity and specificity has been obtained, large quantities of MAbs could be obtained through an in vivo alternative approach known as ascites production, rather than via the in vitro cell culture technique. In ascites production, the MAb-producing hybridoma cells of interest are injected into the abdominal cavity of laboratory mice and then after the next 7 to 14 days, the injected University of Ghana http://ugspace.ug.edu.gh 37 hybridoma cells will secrete an increased quantity of highly concentrated MAbs known as ascites which can be collected. This method of MAb production has some advantages. Firstly, this method is simpler and less time consuming. Also, the abdominal cavity provides an excellent environment for optimal growth of the hybridoma cells because it guarantees a constant temperature, an optimal nutrient and oxygen supply as well as the optimal removal of CO2 and metabolic waste products (Leenaars and Hendriksen, 2005; Hendriksen and de Leeuw 1998). The hybridoma cells in this condition grow to high densities that secrete highly concentrated levels of MAbs (Leenaars and Hendriksen, 2005). In spite of these advantages, the ascites approach for MAb production has fallen into disfavour because of the following reasons. Firstly, the method is considered unethical because of the pain and distress that the mice are probably subjected to (Dewar et al., 2005). Secondly, high-quality in vitro MAb production systems and new production approaches are increasingly progressing and becoming available. In addition, there is substantial evidence of contamination with other immunologically active compounds from the murine peritoneum as well as the risk of contamination of the MAbs with viruses and other microorganisms in the mouse abdominal cavity (Dewar et al., 2005; Lipman et al., 2005). For these mitigating reasons, the studies described in this thesis write-up opted to develop the MAbs by the in vitro tissue culture method rather than via the ascites production approach. Yen Access 2.2.4 Uses and Applications of Monoclonal and Polyclonal Antibodies The importance of the capability of antibodies to selectively bind a specific epitope on a chemical, carbohydrate, protein, or nucleic acid has been demonstrated over the years by several University of Ghana http://ugspace.ug.edu.gh 38 research and clinical applications in which antibodies have been utilized for specific purposes. Such applications include (1) simple qualitative and/or quantitative analyses to determine the presence of a particular antigen in a solution, cell, tissue, or organism; (2) methods to facilitate purification of the antigen, antigen associated molecules, or cells expressing the antigen; and (3) techniques that use antibodies to mediate and/or modulate physiological effects for research, diagnostic, or therapeutic purposes. Antibodies, both MAbs and PoAbs have a variety of academic, medical and commercial uses. It will be impossible to list all the instances in which antibodies have been explored for specific purposes in this discourse. Therefore, the various uses of antibodies which have been outlined below are by no means exhaustive but just to illustrate that antibodies are versatile, and their applicability is limited only by the imagination and determination of the user. The following list therefore, is to indicate how wide the use of antibodies has become in biotechnology. 2.2.4.1 Uses and Application of Antibodies in the Diagnosis of Disease Antibodies are used in several diagnostic tests to detect small amounts of drugs, toxins or hormones. For example, monoclonal antibodies (MAbs) to human chorionic gonadotropin (HCG) are used in pregnancy test kits. Secondly, MAbs and polyclonal antibodies (PoAbs) to Plasmodium antigens are used in rapid diagnostic kits for diagnosis of malaria (Azikiwe et al., 2012; Wongsrichanalai et al., 2007). Also, antibodies are used in ELISA for the diagnosis of AIDS. Finally, antibodies can be used in immuno-localization to detect a particular antigen in a tissue. University of Ghana http://ugspace.ug.edu.gh 39 2.2.4.2 For treatment of disease Antibodies are used in the radioimmunodetection and radioimmunotherapy of cancer, and some new methods can even target only the cell membranes of cancerous cells (Zola and Thomson, 2001). Also, Monoclonal antibodies can be used to treat viral diseases, traditionally considered “untreatable”. 2.2.4.3 Uses and Application of Antibodies in the Classification of Organisms Monoclonal antibodies are used in classification of strains of pathogens. 2.2.4.4 Application of Antibodies for Identification, Monitoring and/or Measurement of Biological Processes Antibodies can be used in conjunction with gel-shift experiments to identify specific DNA-binding proteins (Carey, 2012; Ralston, 2008). Also, antibodies can have a biomarker effects in vivo. These effects enable them to be used in experiments to determine the importance of a particular molecule in vivo, such as the development of the sensory nervous system (Zhu et al., 2013. Finally, Antibodies can also be used in western blotting to detect and/ or identify a protein after separation by SDS-PAGE. With appropriate controls, this procedure can be used to measure the quantity of a protein in response to experimental manipulations. 2.2.4.5 Uses and Application of Antibodies For isolation and purification of proteins Antibodies are used as affinity reagents in the purification and/ or precipitation of proteins from solution (Huse et al., 2002). University of Ghana http://ugspace.ug.edu.gh 40 2.3.0 IMMUNIZATION OF LABORATORY ANIMALS 2.3.1 Introduction Immunization is the introduction of pathogens (disease causing microorganisms) or their proteins into the body of immune-competent organisms with a view to strengthen their immune system so that the receipient organisms would become resistant to the related disease/ diseases. Immunization may be carried out using various mechanisms/ techniques, of which the common approach is vaccination. Vaccination of the body against a particular disease helps to prepare the body’s immune system so that it can fight and prevent the establishment of an infection or disease. For clinical and research purposes the term immunization and vaccination have been used interchangeably. Laboratory (Lab) animals may be vaccinated in order to protect them against diseases. Immunization of lab animals may also be conducted in order to produce antivenoms or antisera for the cure of certain diseases or infections. Lastly, it is widely applied in research either to study the nature of certain infections or to generate antibodies required for serological research. 2.3.2 Route of Immunization of Laboratory Animals for Antibody Production Recently, research on development of protective immunity has been directed toward determining strategies that specifically stimulate heightened and protective immune response. Extensive vaccine and immunization studies of infections with protozoan parasites such as Plasmodium and Leishmania in animal models have been carried out by different strategies involving variation of antigen preparations and the species of animal model used (Leenaars and Hendriksen, 2005; Mohanan et al., 2010). Of the many variables considered in immunization, University of Ghana http://ugspace.ug.edu.gh 41 an important factor that was reckoned to influence the quality (type and strength) of immune response is the route of injection or delivery of the antigen preparation (Mohanan et al., 2010). The route by which an antigen enters the body influences the tissues where immune responses are mounted as well as the magnitude of the immune response elicited (Boa- Amponsem et al., 2001). In line with this therefore, the route of antigen administration has been adjudged to be important in influencing immune responses at the initial site of pathogen invasion where protection is most effective. Furthermore, the choice of injection route is, in turn, shaped to some extent by the choice of the animal species and adjuvant, as well as the nature, concentration, and volume of the antigen preparation to be inoculated (Leenaars and Hendriksen, 2005). For example, the intravenous (IV) route for inoculation of immunogens prepared by emulsifying aqueous antigens in oil-based, large particulate or viscous gel adjuvants such as aluminium salts, Freund’s complete adjuvant (FCA) and Freund’s incomplete adjuvant (FIA) is considered not advisable because of the high tendency (risk) of such antigen preparations to block blood vessels (cause embolism) and kill the animals (Hanly et al., 1995). Also, owing to the fact that injection into any closed space is very painful, the Canadian Council for Animal Care (CCAC, 2002) does not recommend the intradermal (ID) and intramuscular (IM) routes of antigen administration in small rodents like mouse, rat, and hamster, because these small animals do not have enough spaces in their muscle and skin (CCAC, 2002). According CCAC (2002), therefore, the following four guidelines should be kept in mind when considering the route of antigen administration. They are:  Inject the smallest possible volume per site.  Use multiple injection sites when necessary. University of Ghana http://ugspace.ug.edu.gh 42  Space injection sites far enough apart to avoid coalescing of inflammatory lesions. Failure to do so may result in significant tissue necrosis.  No single route of administration is ideal In line with the above mentioned recommendations therefore, the most frequently used routes of injection of large animals such as rabbit, goat and sheep for PAb production are subcutaneous (SC), ID, IM, intraperitoneal (IP), and IV (Leenaars and Hendriksen, 2005). As all the routes of immunization have both advantages and demerits, no single route is considered perfect as far as the survival of the host organism and the elicitation of immune response are concerned (Shimizu, 2004). Having satisfied all these conditions, the best route of antigen administration to adopt is the type that can yield heighten immune response and antibody production, with minimal or no inflammation and/ or death of the host animal (FSUIRAP, 2013). Through extensive immunization research, FSUIRAP (2013) has documented the merits and demerits of the various routes of antigen administration. This report and studies conducted by other researchers on the routes of antigen administration (Jerusalem and Eling 1969; Shimizu, 2004; Leenaars and Hendriksen, 2005; (FSUIRAP, 2013) have shown that the four major routes, IV, IP, SC and IM routes have been used most extensively in the immunization of mice towards the production of antibodies. 2.3.2.1 Intramuscular Route of Immunization This route of immunization is often relied upon to provide a safer (safer than the intravenous route) rapid uptake into the bloodstream and lymphatic system. However, this is dependent upon the size of the antigen. It is a good site for small molecular weight drugs that are irritant as they University of Ghana http://ugspace.ug.edu.gh 43 are rapidly absorbed into the blood and the inflammatory response distributed. Large molecules are more likely to be absorbed primarily by the lymphatic system which lie only in the fascial planes. The advantage in this route of inoculation is that larger amounts can be injected intramuscularly. The disadvantage is the more prolonged absorption and spread of antigen- adjuvant preparation along the fascial planes. Such spread (and accompanying inflammation) may cause problems especially where nerves are encountered. In addition, because of the prolonged assimilation of the immunogen, it takes a long time to elicit immune response which are normally poor (Jerusalem and Eling, 1969). Lastly, the use of this route for immunization of animals with small muscle masses is discouraged and therefore, not generally recommended in rodents because of their limited muscle mass (FSUIRAP, 2013; Leenaars and Hendriksen, 2005). However, the use of CFA via this route is controversial. Intramuscular injections are usually made in the biceps femoris or quadriceps muscle mass. A great caution is required to avoid adjacent nerves and blood vessels as well as facial planes when injecting into a muscle bundle. The intramuscular route of immunization was not therefore used in this study partly because of the foregoing reasons. Also, the BALB/c mouse muscle does not have enough space to accommodate the amount of immunogen required to elicit the heighten degree of immune response necessary to produce antibodies with high sensitivities as required and was not therefore considered suitable for this study. In addition, considering the number of immunizations, both primary and secondary, that were carried out on each mouse, the intense pain and the tendency to immobilize the mice and prevent them from feeding normally, which is against CCAC (2002) recommendations, and would have also caused the premature death of the mice before immune response. Finally, in a study conducted by Jerusalem and Eling (1969) to investigate the effect of the three routes of antigen administration on immune response University of Ghana http://ugspace.ug.edu.gh 44 of BALB/c mice to Plasmodium antigens, it was reported that mice immunized via the IM route elicited the lowest degree of immune response in terms of antibody titre, sensitivity, specificity and protection against challenge infection with live Plasmodium parasites, which is an indication that the use of the IM route could lead to the generation of antibodies with low sensitivity and specificity. It is for these reasons therefore, that the intramuscular route was not used for immunization of mice in this study. 2.3.2.2 Subcutaneous Route of Immunization The subcutaneous (SC) route of antigen administration is probably the most frequently used route (FSUIRAP, 2013) because it is one of the easiest and rarely painful injection technique to carry out (Shimizu, 2004). Owing to its painless nature, it is conducted usually when the mice are conscious as for the intraperitoneal (IP) route (Shimizu, 2004). Up to 3ml of antigen preparation are recommended to be administered per site of this route of injection (Shimizu, 2004). As the antigen is introduced mainly via the lymphatic system (FSUIRAP, 2013), the rate of absorption is slower than the cases for the IP and IM injections (Simmons and Brick, 1970). The rate of absorption is dependent upon the blood flow in the area (dependent upon skin temperature), activity of underlying muscles and contact area (Shimizu, 2004). The loose skin areas will allow further spread of the antigen-adjuvant preparation, thereby increasing the contact area. It is a good choice for antigen administration. It is likely to induce anaphylactic reaction if absorbed into the bloodstream. The disadvantage associated with this technique is that inflammatory reactions are less likely to stay at the site of injection but may migrate with the development of fistulous tracts (FSUIRAP, 2013). Also, the relatively slower rate of absorption University of Ghana http://ugspace.ug.edu.gh 45 of the immunogen via this route than the IP route make the latter superior in terms of the quality of immune response. Subcutaneous administrations are made into the loose skin over the interscapular area as follows. The mouse is manually restrained and then placed on a clean towel or solid surface. The needle is inserted under the skin of the interscapular or inguinal area, and while being tented by the thumb and forefinger, the immunogen is then inoculated. The SC route was not employed for immunization of mice in this study because of its associated disadvantages of weaker immune response and migratory inflammatory reactions which could have a negative impacts on the survival of the mice through the immunization period and the sensitivity of the antibodies produced. Furthermore, the SC route was not used because, like the IM route it is not recommended for the immunization of rodents (FSUIRAP, 2013; CCAC, 2002). 2.3.2.3 Intravenous Route of Immunization The intravenous (IV) route of administration delivers antigen to the spleen and secondarily, to the lymph nodes. It is a good choice for administration of soluble antigens without any adjuvant. It is not recommended for antigens prepared in adjuvants due to the risk of embolism, as mentioned above. Adjuvants that may be used intravenously include liposomes, properly prepared water-in-oil double emulsions and dispersed alum. The disadvantages of this route include 1) lack of antigen depot effect, 2) poorer response in antibody titre when used as the route for primary immunization, 3) increased risk of tolerance or anaphylactic reactions, 4) it is the most lethal of all the routes of immunization (Jerusalem and Eling, 1969), 5) it requires high technical expertise and skill to perform (FSUIRAP, 2013; Shimizu, 2004; Jerusalem and University of Ghana http://ugspace.ug.edu.gh 46 Eling, 1969). It is often used for booster injections following primary immunization with an antigen-adjuvant given via another route. In spite of the above, IV injection has certain advantages over other routes. Highly concentrated or irritating solutions, or solutions with high or low pH can be administered intravenously provided that the rate of injection is kept slow and precautions are taken to avoid getting the solution outside the vein. Compounds that are poorly absorbed by the digestive tract may be given intravenously. The intravenous (IV) route was not used in this study because 1) It is the most fatal (Jerusalem and Eling, 1969) among the four routes of immunization mentioned above; 2) The laboratory animal of choice for this study was the BALB/c mice, which were chosen based upon the cost of purchase and maintenance of the animal, scarcity of space and that the myeloma (NS1, P3X63Ag8.653 [Ag8],) cell lines available for cell fusion and production of the monoclonal antibodies originated from mice. 2.3.2.4 Intraperitoneal Route of Immunization The intraperitoneal (IP) route is most frequently used for immunization of rodents, less often in other species (Shimizu, 2004; FSUIRAP, 2013). Antigen is taken up by the lymphatic system rapidly and transferred to draining nodes, the thoracic duct and hence the vascular system. The advantages of this route of immunization include the relatively larger volume of preparation that can be injected, several different types of adjuvants can be used and the antigen is widely distributed to lymphoid tissue. Also, unlike the intravenous route, injection through the (IP) route can lead to the elicitation of heighten immune response and protection which are slightly lower but comparable to that induced by the intravenous route (Bhowmick et al., 2009; Jerusalem and Eling, 1969). In addition it is relatively far safer than the inoculation via the intravenous route University of Ghana http://ugspace.ug.edu.gh 47 (Jerusalem and Eling, 1969). The disadvantage of this route for administration of antigen, however, is the risk of anaphylactic shock if boosters are rapidly absorbed into the vascular system. Also, owing to the fact that the use of complete Freund’s adjuvant (CFA) through the IP route to produce polyclonal antibodies induces inflammation, peritonitis, and behavioral changes (CCAC 2002) it is adviced that it should be carried out only after scientific justification and approval (CCAC, 2002). Finally, the IP route is not recommended in larger animals such as rabbit, goats and above (Hendriksen and Hau 2003). The production of rodent peritoneal exudate by the intraperitoneal administration of antigen and adjuvant is a widely recognized valid scientific procedure for obtaining high-titer reagent. Undesirable side effects of painful abdominal distention and the resulting distress can be avoided by daily monitoring and relief of ascites pressure, or termination of the experiment. This route of immunization is the most commonly applied because it is technically simple and easy. It allows quite faster but long enough periods of absorption from the repository site. The rate of absorption by this route is usually one-half to one-fourth as rapid as from the intravenous one (Woodard, 1965). The limitations of this route are few. Firstly, the affected tissues may react adversely to irritating substances. Also, the affected tissues are often less tolerant to solutions of non-physiological pH, as this route requires the use of isotonic solutions of which quite large volumes can be administered. This route of immunization was therefore used in this study because it is relatively safer, it results in the elicitation of a heighten immune response and that it is the most widely used method for immunization of BALB/c mice to generate antibodies. University of Ghana http://ugspace.ug.edu.gh 48 2.4.0 IMMUNITY TO MALARIA 2.4.1 Innate Immunity to Malaria In malaria endemic areas, immunity to malaria begins as natural defense or innate immunity, which is observed, right from birth, in populations continually exposed to malaria parasites. The first example is observed in people with inherited conditions such as sickle cell anaemia, beta-thalassaemia and glucose-6-phosphate dehydrogenase deficiency (retrieved from http://malaria.wellcome.ac.uk/ doc_WTD023885.html on April 2, 2007). These conditions are common in people from malaria endemic communities, and are known to cause deformities in red blood cells (RBCs), which make it difficult for the RBCs to be infected by malaria parasites and other pathogenic organisms. The second example of innate immunity to malaria is demonstrated in people of west African origin, who are known to have RBCs that lack proteins called Duffy antigens on their surfaces (Weatherall et al., 2002;). These proteins act as receptors for P. vivax merozoites during infection of the RBCs, so in people without the Duffy antigens, the merozoites cannot bind onto the RBCs, and are cleared during the blood stage of infection, thus making these individuals resistant to infection by the P. vivax malaria parasite (Weatherall et al., 2002). Lastly, infants from birth to about three months or even up to about 12 months of age have some protection against malaria which is conferred by immunoglobulin G (IgG) antibodies that crossed the placenta from mother to child during pregnancy (Moormann, 2009). This immunity, however, begins to decline from about the third month to about one year of age when it is completely lost as a result of loss of the maternally acquired IgG antibodies (Moormann, 2009). University of Ghana http://ugspace.ug.edu.gh 49 2.4.2 Acquired Immunity to Malaria The infection of immunocompetent humans by malaria leads to development of acquired immunity, such that individuals that are repeatedly exposed to P. falciparum infection acquire partial immunity. The process of acquired immunity begins in babies and infants (with a few exceptions) who are exposed to Plasmodium parasite infection (malaria), on a regular basis, and is sustained into later life (Phillips, 2001). Most of these young people survive infection with P. falciparum, however, in about 1 to 2% of the infected individuals, severe malaria develops and could be fatal. Although children develop protective immunity against P. falciparum, such protection is not easily acquired (Phillips, 1994). In areas of stable or high malaria transmission and endemicity, the pattern is for non-immune children (babies and infants) up to 5 years of age to be frequently infected by Plasmodium parasites early in life (Weatherall et al., 2002). Often, a number of such children do experience more severe malarial symptoms, and become at risk of dying from the disease, while adults suffer fewer clinical malaria episodes (Chattopadhyay et al., 2003). However, as immunity develops with repeated infection, parasite prevalence, density, number of clinical episodes and the severity of the disease decline progressively with increasing age (Weatherall et al., 2002). In non-immune adults however, effective immunity probably develops more quickly after first infections (Baird, et al., 1991; Baird, 1995). For instance, it was observed in a malaria endemic area in Indonesia that after 2 years, there were no fatalities among adult residents, who were originally non-immune and had migrated from malaria non-endemic areas. This suggested that lifesaving immunity developed in 2 years in adults compared with 5 years in small children (Baird, 1995). University of Ghana http://ugspace.ug.edu.gh 50 The common thinking is that natural acquired immunity to malaria is normally strain- specific, and appears to be lost if a person migrates from an endemic to a non endemic area for an extended period of time, suggesting that repeated exposure is necessary to maintain resistance (McGregor, 1986; retrieved on April 2, 2007, from http://malaria.wellcome.ac.uk/ doc_WTD023885.html). Immunity to malaria is never complete, even in adulthood and during the transmission season, clinical episodes do occur and parasites may be observed in blood films in the absence of clinical signs. Owing to the fact that immunity is apparently invariably incomplete, a vaccine that will confer total protection, therefore, requires a level of immunity never achieved by natural exposure. If natural immunity could be mimicked, then vaccination would prevent severe malaria and malaria-related deaths, but would not give complete protection (Weatherall et al., 2002). 2.4.3 Vaccines against Malaria-1 The unacceptable morbidity and mortality (Hill, 2011) caused by malaria to humans have led to the deployment of numerous control measures, including chemotherapy and destruction of vector mosquitoes by insecticides (Graham et al., 2004) as well as management of the environment to discourage the proliferation of vector mosquitoes (Randell et al., 2010). Nevertheless, the destructive toll of this disease has not seen any satisfactory improvement over the past decade. (WHO fact sheet 2002; WHO World Malaria Report, 2008; Snow et al., 2005). Acknowledging the phenomenal success of vaccines against diseases such as poliomyelitis, measles, diphtheria, tetanus, rabies, and more particularly, their ability to completely eradicate smallpox (Sallusto et al., 2010), that used to kill and disfigure a substantial population of the world (Breman and Arita, 1980), coupled with the belief in the potential of vaccines to reduce the global burden of infectious University of Ghana http://ugspace.ug.edu.gh 51 diseases, the foregoing hopeless nature of malaria and its existing control strategies, mainly in endemic populations, led to the conclusion that vaccination against P. falciparum and P. vivax is the method of intervention with the greatest promise to reduce the morbidity and mortality associated with severe malaria in areas of intense transmission (Miller and Hoffman, 1998) and even would contribute to eradicating the disease with a very high probability (Greenwood, 1997). In addition, with the success of early vector control programs suggesting that it is reasonable to hold an optimistic view (Phillips, 2001), the belief in the ability of vaccines to eradicate malaria resulted to a variety of sustained efforts to develop effective malaria vaccines (Hill, 2011) to control the disease. Immunological memory is the ability of the immune system to respond with greater intensity to infection when the causative pathogen is encountered again, and constitutes the basis for vaccination (Ahmed and Gray, 1996). To date, vaccination remains the most effective method of preventing infectious diseases, and represents the most relevant contribution of immunology to human health (Plotkin and Plotkin, 2008; Siegrist, 2008). Notwithstanding the tremendous achievement of vaccines against the viral and bacterial diseases mentioned above, and the remarkable optimism that they could be used to eradicate vector bone diseases, especially malaria, development of vaccine against this disease represents a major challenge, even with unlimited resources to devote to the task because, firstly, it would be a very tall order to produce a vaccine against a parasite for which limited natural exposure does not stimulate a protective immune response (Phillips, 2001). Also, there are a number of other features of malaria parasites which make the development of vaccine against the disease particularly difficult (Phillips, 2001). For example, according to Phillips (2001) Protozoa are relatively complex, and in malaria parasites, it is estimated that there are 5,000 to 7,000 proteins. Immunity to each stage of the life University of Ghana http://ugspace.ug.edu.gh 52 cycle is largely specific, probably because each stage produces its own novel antigens. Therefore, selecting targets for vaccine-induced immunity and the corresponding peptides with which to induce that immunity has been extremely difficult. Also, Phillips (2001) reported that unlike most viral vaccines, malaria vaccines must virtually be subunit vaccines because, although it is possible to grow all stages of the parasite life cycle in vitro to produce a whole-organism vaccine it is presently difficult to generate large quantities of sterile, efficacious protein material that is required for mass production of whole-parasite vaccines (Phillips, 2001; Draper, 2012). In spite of the the suggestion by Phillips (2001) that malaria vaccines must be subunit vaccines, Wykes and Good (2007) stated that evidence from in-depth studies of the homologues of the Plasmodium vaccine candidate antigens show that these individual proteins on their own have various inherent immunological characteristics that make it difficult, if not impossible to use them in development of effective sub-unit vaccines against malaria. 2.5.0 DIAGNOSIS OF MALARIA Diagnosis is a medical term used to indicate the act of demonstrating the presence of disease. It is probably the most important component of the healthcare delivery system, and has various ramifications depending on the nature of the disease as well as the geographic, social, cultural and the economic factors that influence its development and application. Prompt, accurate and safe diagnosis is the key to effective disease management, a major intervention measure of the Global Malaria Control Strategy (WHO, 1993). This is probably because early and accurate diagnosis is crucial in the development of management strategies for the prevention of diseases. Also, at the onset of severe and complicated malaria, timely and University of Ghana http://ugspace.ug.edu.gh 53 accurate diagnosis is essential to prevent death. Nevertheless, it is of great concern that poor diagnosis continues to hinder effective control of malaria (WHO, 2000). Several approaches to the diagnosis of malaria are currently in adoption. Each approach has characteristics, including costs, availability, and ease of performance and accuracy that determines its applicability to different situations (WHO, 2000). 2.5.1 Clinical Diagnosis of Malaria The clinical method is presently the first-line and the most widely used approach to malaria diagnosis. It is based on the observation of clinical signs, the most prominent being fever {persistent abnormal increase in body temperature (temp.), i.e. axillary temp. ≥37.3oC; oral temp. ≥37.8oC; rectal/ ear temp. ≥38.3oC} accompanied by headache, chills, perspiration and anorexia (loss of appetite). It is inexpensive and requires no special equipment or supplies. It has been the only feasible diagnostic approach that residents, particularly, of rural endemic areas are familiar with, and frequently utilize in self-diagnosis of malaria (WHO, 2000). Nevertheless, clinical diagnosis is presumptive and unreliable because, malaria presents non-specific symptoms, which overlap with those of other febrile diseases such as meningitis, typhoid fever, influenza, septicaemia, hepatitis, scrub, gastro-enteritis viral encephalitis as well as haemorrhagic fevers (WHO, 2000; White, 1998; Bradley, 1996). Also, when chloroquine and other low-cost and less toxic drugs were highly effective, especially, in areas of high malaria transmission where microscopy is not readily available, treatment of malaria based on clinical signs alone was generally considered cost-effective and justifiable (WHO, 2000; Azikiwe et al., 2012). In areas of low-malaria transmission, however, indiscriminate dispensing of anti-malarial drugs due to unconfirmed clinical diagnosis has led to the emergence of multi-drug resistant malaria which University of Ghana http://ugspace.ug.edu.gh 54 requires more expensive and toxic mefloquine or artemisinin-based combination therapies. It is therefore important to minimize anti-malarial drug use based on presumptive clinical diagnosis alone. 2.5.2 Microscopic Diagnosis of Malaria Conventional light microscopy is presently the established and most acceptable method for confirmation of Plasmodium infections (Katzin et al., 1991; Payne, 1988). Careful examination of Giemsa, Wright’s or Field’s -stained thick and thin blood smears, well prepared from finger-prick or venous blood samples by an expert microscopist is presently the “gold standard” for specific detection and identification of malaria parasites (WHO, 2000). Microscopy is also considered the gold standard for determining the efficacy of antimalaria drugs or vaccines (Ohrt et al., 2002), and the yardstick for evaluating the sensitivity and specificity of new diagnostic methods (Steenkeste et al., 2010; Okell et al., 2009). Microscopic diagnosis of malaria has several advantages. It is relatively sensitive and can detect 5-10 parasites per a microlitre of blood when carefully done by a skilled technician (WHO, 1990). However, under general field conditions the detective capability might be more realistically placed at 100 parasites per a microlitre of blood (WHO, 1988). Microscopy is very specific and highly informative. It allows parasites to be distinguished into species such as P. falciparum, P. malariae, P. vivax, P. ovale or P. knowlesi and circulating stages such as trophozoites, schizonts or gametocytes (WHO, 2000). Also, microscopy enables quantification of parasites per leukocytes or erythrocytes that is needed for determination of parasitaemia or assessment of parasitological response to chemotherapy (WHO, 2000). The blood smear preparations can provide a relatively permanent record of diagnostic findings and be subject to University of Ghana http://ugspace.ug.edu.gh 55 quality control (Payne, 1988). Furthermore, microscopy is relatively inexpensive, with cost estimates for endemic countries ranging from US$0.12 to US$0.40 per slide examined (Palmer et al., 1998). The cost however decreases when large numbers of samples are examined, and also when shared with other disease control programmes (WHO, 2000). Nevertheless, microscopy has several disadvantages. It is labour-intensive and time- consuming, normally requiring at least 45 minutes to yield results (WHO, 2000). It is exacting and requires highly motivated skilled or well-trained and supervised technicians with good microscopes, reagents and electricity. Unfortunately, these conditions are usually not available at the peripheral levels of the health care delivery system (WHO, 2000) where most deaths and morbidity occur. In addition, long delays in communicating the microscopic results to clinicians, make decision on treatment to be taken without the benefits of the results. In view of this, Payne (1988) indicated that microscopic diagnosis of malaria using scarce resources could be highly doubtful and unreliable. Furthermore, microscopic method of diagnosing malaria is not safe enough because it is invasive and exposes healthcare workers to blood. Blood is used in traditional societies for atonement and regarded as the essence of life. It is used to appease the angry spirits, ancestors and gods, or offered to thank the gods and ancestors for their protection in the past, present and future. The Efutus of Winneba in Ghana, for example, offer blood to their god “Penyi Otu” during Aboakyir festival as a thanksgiving sacrifice (Otabil 1992). Blood is also seen as an important ingredient in the preparation of very potent traditional medicine and magic. On account of these, blood collection in most of the highly malaria endemic traditional African societies is associated with fetishism, and therefore, making it difficult to encourage the use of diagnostic tests that involve invasive procedures at the community and University of Ghana http://ugspace.ug.edu.gh 56 household levels, which is an important strategy for reduction of malaria morbidity and mortality in traditional African societies(WHO, 2004). 2.5.2.1 The Roles of Thick and Thin Blood Smears in Diagnosis of Malaria Blood obtained by pricking a finger or an earlobe is an ideal sample, because these tissues are rich in capillaries, which have very high concentrations of developed trophozoites or schizonts (Moody, 2002). Alternatively, blood obtained by venipuncture collected in heparin or sequestrine (EDTA) anticoagulant-coated tubes may be suitable if processed immediately, to prevent alteration in the morphology of malaria parasites and white blood cells (Moody, 2002). The thick blood film concentrates red blood cell (RBC) layers over a small surface area by 20 to 30 fold to be stained as an unfixed preparation using Field’s stain or diluted Giemsa or Wrights stain (Moody, 2002). This characteristic enhances the sensitivity of the blood film technique for detection of low-level parasitaemia and reappearance of circulating parasites during infection, recrudescence or relapse. Lysis of the RBCs during staining can make examination for parasites more difficult until experience is gained in finding the parasites among the white blood cells (WBCs) and platelets. The thin blood film on the other hand, comprises a monolayer of RBC that is fixed with methanol and stained with diluted Giemsa or Wright’s stain using buffered water at pH 7.2 to distinguish parasite inclusions in the RBC. The fixed monolayer of RBC in this procedure makes it more specific because, morphological identification of parasites to the species level is much easier than the thick blood film examination. Also, the thin blood film makes it easier to see and count parasites so it is mostly the choice for routine estimation of parasitaemia. The ability to count parasites in sequential blood films is required in monitoring the response to therapy, especially, for Plasmodium falciparum infections. University of Ghana http://ugspace.ug.edu.gh 57 2.5.3 Rapid Diagnostic Tests for Malaria Conventional diagnosis still uses the skilled but laborious and time-consuming microscopic examination of thin and thick blood films stained with Giemsa’s or Field’s stain. Newly developed tests include the quantitative buffy coat method (Becton Dickinson, Sparks, Md.) for the fluorescent staining of parasites after concentration (reported to be as good as thick films for P. falciparum but not for the other species (Baird et al., 1992); the CareStat MalariaTM (AccessBio Inc., New Jersey, USA) (Eibach et al., 2013) and the Paracheck (Orchid Pharmaceuticals) (Reyburn, et al., 2007), which are based on the immunological capture of P. falciparum histidine-rich protein 2 and/ or parasite pan-specific lactate dehydrogenase or aldolase enzymes in whole blood (Azikiwe et al., 2012; Moody, 2002). These antibody-based dipstick tests are still being evaluated. PCR-based diagnostic tests for human malarias have been developed (Morgan et al., 1998; Eibach et al., 2013), but these are more applicable to large-scale surveys than to clinical diagnosis. PCR has been especially effective at detecting submicroscopic levels of parasitemia (Alemu et al., 2014; Mahajan et al., 2012; Okell et al., 2009; WHO, 2000). The quatitative buffy coat (QBC) test is a microscopy- based test that employs a fluorescent dye to enhance the visibility of parasites in microcapillary collected blood samples. Even though it is rapid and takes about 15 mins to perform it is invasive and requires blood to perform. Also, the QBC system comprising, the special micro capillary tubes with stoppers, microcentrifuge, the QBC reader and fluorescent microscope which must be powered by electricity are very expensive and cannot be afforded by hard to reach rural communities in which morbidity and mortality are highest. In view of this very few specialized health centers have and utilize the QBC system in the diagnosis of the disease. University of Ghana http://ugspace.ug.edu.gh 58 2.6.0 MALARIA TRANSMISSION AND ITS EFFECT ON AGE-DEPENDENT DISEASE BURDEN IN ENDEMIC POPULATIONS The pathogenesis of malaria is such that individuals with no or limited resistance to the disease, tend to be at the greatest risk of infection, harbour the greatest parasite load and suffer from the severest outcomes of the disease (Stauffer and Fischer, 2003). Humans, right from birth to about 6 months after birth, have maternally transmitted anti- malaria IgG antibodies within them which give them immune protection against malaria (Doolan et al, 2009). After 6 months of age and onwards, the maternally acquired antibodies and the corresponding immunity to diseases begin to wane, while the body builds up its own acquired immunity for protection against subsequent infections (Doolan et al, 2009). During the time of development of adequate acquired immunity, it is common for unimmune individuals to suffer from the worst outcomes of infection, including heavy parasite density, heavy disease burden and high rate of mortality, which decline as the individual grows older and continuously build up adequate protective immunity (Doolan et al, 2009). In an area of high transmission intensity, it takes about 5 years on the average (Doolan et al, 2009), for unimmune children to develop the theoretical immunity necessary to confer partial protection against the worst outcomes of malaria infection (Doolan et al, 2009), so the severest outcomes of the disease including heavy parasite density, is concentrated in children under 5 years of age (Doolan et al, 2009). However, in an area of low transmission intensity, the gestation period for acquisition of adequate protective immunity against malaria takes longer than 5 years (Wipasa et al., 2002), and could even extend into adulthood, because the amount of infectious Plasmodium antigens that characterize low transmission of parasites is not enough to induce development of immune protection within 5 years. University of Ghana http://ugspace.ug.edu.gh 59 2.6.1 Malaria Transmission at Kpone-on-Sea Owing to the devastating nature of malaria, efforts are being made worldwide to control the disease (Reviewed by Tchouassi et al., 2012). As part of this worldwide malaria control effort, Kpone-on-Sea (KOS), a malaria endemic coastal fishing village in southeastern Ghana, is being developed as a model site for malaria intervention studies with a view to control the disease in the endemic population. The strategies being adopted include improved vector control, chemotherapy, use of insecticide treated bed net (ITN) and possibly, vaccination in the foreseeable future (Tchouassi et al., 2012). For the vector control strategy to be effective there was the need to characterize the malaria transmission pattern of the area. In a study to characterize malaria transmission at KOS (Quakyi et al., 2004), and at about the same period that data for the studies reported in this thesis was collected, the intensity of malaria transmission at KOS was estimated to be variable with an entomological parasite inoculation rate, EIR = 62.1 (Tchouassi et al., 2012). A variable or an unstable malaria transmission intensity for KOS, implies that children in this community, after birth, can take 5 years or longer to acquire adequate protective immunity necessary to mitigate some of the adverse outcomes of malaria infection. Also, between 2004 and 2008, a WHO funded project, the KOS Malaria Project, carried out a multidisciplinary case control study of malaria pathogensis and immunity in souththern Ghana, during which artesunate- amodiaquine was used to treat infected members of the Kpone community (Quakyi et al., 2004). Furthermore, the Ghana Health Service embarked on the distribution of insecticide treated bednets (ITN) for use by pregnant women and nursing mothers in a bid to control malaria transmission at KOS. It is highly probable therefore, that these intervention programmes together, would lead to a considerable reduction in malaria transmission at KOS, especially, among University of Ghana http://ugspace.ug.edu.gh 60 children under age 5 who shared the ITNs with their parents and had less exposure to infectious mosquito bites. The overall effect conceivable, is that the period for development of protective immunity would be shifted from 5 years on the average to a longer one, probably, 10 years, 15 years (Wipasa et al., 2002) or even longer, which in turn would lead to the shifting of the risk of the severest malarial outcomes into the 6 to 10 years age group of individuals (Griffin et al., 2014; Schellenberg et al., 2004). In view of this, after age 5, when the children are no longer allowed to sleep under ITN, and are exposed to infectious mosquito bites, it is probable that they would be incapable to offer any resistance to malaria infection and consequently would experience the worst outcomes of the disease including harboring of the heaviest load of parasites. University of Ghana http://ugspace.ug.edu.gh 61 CHARPTER 3 GENERAL MATERIALS AND METHODS 3.1.0 STUDY SITE 3.1.1 Kpone-on-Sea Kpone-on-Sea is located in the Dangme West District within the Greater Accra Region of Ghana (Map 1). It is situated on 5o69'N, 0o06'E within the coastal savanna belt of West Africa. The village is surrounded by coastal scrub. It is bordered, on the east by Prampram, on the west by Tema, on the North by the industrial free zone, and on the south by the gulf of Guinea which is 139 metres away. Kpone stands at an altitude of 50m to 100m above sea level and has an equatorial climate. Temperatures range from 24.4oC to 27.8oC with a mean of 26.1oC. Over the years maximal transmission of malaria occurs during the major rains (May to July) and after the minor rains (November). Mean annual rainfall averages between 1,133 and 3,606 mm with an average relative humidity index ranging from 78% to 85%. The land formation and the drainage patterns of the 4 sectors of the village were such that all water from the village drains into a stream that lies on the outskirts of Kpone village. A lagoon also on the outskirts of the village separates Kpone from Prampram. Kpone has limited standing pools of stagnant water to promote mosquito breeding. The estimated population of Kpone was 9,300 in 2004 (Quakyi et al., 2004). Majority of the residents (80%) were of the Ga and Ga- Dangbe ethnic groups. Most of the inhabitants were fisherman of whom many were also involved in farming. The Dangbe West District Health Directorate manages health service delivery at Kpone. University of Ghana http://ugspace.ug.edu.gh 62 3.2.0 STUDY DESIGN The proposed research involved subjects from KOS. Finger-prick blood samples from study subjects were examined microscopically for Plasmodium infection and parasitaemia. Urine samples were also collected from study subjects and examined for biomarkers of malaria such as glucose, ketones, Plasmodium falciparum histidine rich protein II (pfHRPII) and hepcidine. Malaria antigens were then extracted from the urine of malaria positive individuals for monoclonal antibody (MAbs) generation. Plasmodium falciparum parasites were cultured in vitro and used to prepare crude parasite antigens. These antigens together with the urinary Plasmodium proteins emulsified in Freund’s complete adjuvant (FCA), were used to initiate immunization of laboratory bred BALB/c mice to select antibodies that target P. falciparum antigens. After the third booster post primary immunization, the P. falciparum antigens prepared from urine of positive subjects and cultured parasites emulsified with Freund’s incomplete adjuvant (FIA), were then used to do the final booster of immunizations prior to cell fusion. Spleen cells from immunized BALB/c mice were used to generate anti- P. falciparum species-specific MAbs, and explored to develop a urine-based membrane-based dipstick and micro-plate ELISAs for detection of parasite antigens in the urine of infected individuals. The accuracy of the developed urine-based dipstick-ELISAs was evaluated in the study areas and hospitals by comparing its sensitivity and specificity with those of ICT and microscopy. 3.2.1 Sample Size Determination For accuracy, the developed urine-based dipstick assay has to be evaluated for sensitivity and specificity which requires collection of adequate number of samples. The following therefore University of Ghana http://ugspace.ug.edu.gh 63 describes the estimation of the number of samples required for evaluation of the developed urine- based dipstick test. In 2004, the estimated population size of Kpone-on-Sea (KOS) was 9300 (Quakyi et al., 2004). In 2005, the prevalence of malaria among patients visiting the Kpone Health Centre as determined by a pilot study was 52.0% (0.52), McKakpo et al. (2006, data unpublished). For the proposed urine based dipstick test (RUBDA) to be as accurate as required, it was expected to have a sensitivity of at least 96% (0.96). Considering that the lower 95% confidence limit for the test should not fall below 85%, then from the sample size determination chart for diagnostic assays provided by Flahault et al.( 2005), 625 controls were required for evaluation of accuracy of the RUBDA. However, the number of controls required for a diagnostic test in a population of disease prevalence greater than 50% (Prev > 0.50) is related to the number of cases and prevalence by the equation: Neon = Ncas [(1-Prev) / Prev)] (1); Where, “Ncon” and “Ncas” are the required number of controls and cases, respectively, and “Prev” is the prevalence of the disease in the community. Therefore from equation (1), 625 estimated controls will require 677 cases or a total of 1302 subjects for evaluation of the diagnostic test. Convenient sampling was used to recruit patients for the study. This was necessary to obtain adequate number of infected samples before the end of the rainy season. The rainy season is the most favourable period for obtainment of the maximum number of Plasmodium-infected individuals from the Kpone study community. The development of the urine-based dipstick test for malaria required Plasmodium proteins isolated from malaria infected urine samples which are University of Ghana http://ugspace.ug.edu.gh 64 obtainable mostly at the peak of the rainy season. Since house to house collection of urine samples would have taken a longer time which would have extended far beyond the rainy season into the dry season, sample collection was done only at the health centre instead. 3.2.2 In Vitro Propagation of Laboratory (3D7) Strain of Plasmodium falciparum Parasites Citrate Phosphate Dextrose (CPD) anti-coagulated venous blood was collected from healthy individuals and kept for 24 hrs at 4oC. The anti-coagulated blood samples were then washed 5 times in prewarmed (37oC) washing medium and at each washing step samples were centrifuged for 7 minutes at 738 x g (2000rpm) to obtain fresh RBCs. Buffy coats were removed after each centrifugation.The 3D7 laboratory strain parasites were cultured in canted neck flasks until their maturation to schizont stage, using the Trager and Jensen protocols (Trager and Jensen, 1976 and 2005) for continuous culture of human malaria parasites with slight modification. Cryopreserved 3D7 strain parasites in RBCs in vials retrieved from a liquid nitrogen tank were thawed in water bath at 37oC. The cells were centrifuged at 738 x g (2000 rpm) for 5 minutes and the supernatant discarded. Equal volume of thawing mix (3.5% NaCl in distilled water) was added to lyse the RBCs and release parasites from the erythrocytes. The parasites were washed twice with 1ml of complete culture medium (RPMI-1640, supplemented with gentamycin, Albumax and L-Glutamine) and then added to a culture flask (25ml canted neck flask) containing 5ml complete parasite medium and 200µl of packed uninfected O+, sickling negative erythrocytes. The flask was flushed for 30 sec with gas mixture (2.0% O2, 5.5% CO2 and 92.5% N2) at 1.5-2.0 bar pressure and incubated at 37oC to provide a suitable condition for parasite growth. The culture medium was changed daily to maintain the culture until a high yield of parasite growth at the schizont stage was obtained. University of Ghana http://ugspace.ug.edu.gh 65 The parasitaemia of the culture was obtained using microscopic examination of geimsa stained thin films, and the percentage parasitaemia was determined using the formula indicated below; Number of infected erythrocytes X 100 Total number of erythrocytes (500 erythrocytes) When percentage parasiteamia exceeded 5%, the culture was transferred into a bigger flask (75ml canted neck flask). Added to the flask was 25ml culture medium and 800l of fresh RBC’s (to reduce the total percentage parasiteamia to 1%), gassed and then kept in the incubator at 37oC. The culture was continuously maintained until percentage parasiteamia of between 8- 10% was obtained with the parasites mostly in their schizont stage. At this point, the culture was used for the preparation of crude malaria antigen by first harvesting the schizonts using the magnetic activated cell sorting technique and then extracting the antigen, using the method explained below. 3.2.3 Extraction of Soluble Crude Plasmodium falciparum Antigens from In Vitro Cultured 3D7 Parasites The schizont stage parasites were isolated and antigens prepared. In this process, RBCs infected with the schizont stage parasites were separated from the uninfected RBCs and ring stage RBCs using Magnetic-Activated Cell Sorting (MACS) technique. The MACS column was mounted within the strong magnetic field which was suspended from a glass platform. The column was run with a PBS buffer to remove cellular debris. A 21G (0.8 x 0.4mm) needle was then fixed to the base of the column to regulate flow University of Ghana http://ugspace.ug.edu.gh 66 rate from the column. The column was then washed twice with 2.5ml washing medium (RPMI- 1640, L-gluthamine and gentamycin) The parasite cultures were diluted with phosphate buffered saline (PBS, pH 7.2) to cause disintegration between both the infected and uninfected RBCs for a successful purification resulting in a good yield. The diluted parasite culture was then passed through the column and thereafter washed with a washing medium until colour of the eluent changed from red (culture medium) to colourless. As a result of their high haemozoin content which is paramagnetic, parasites in the late developmental stages (mainly schizonts) bound to iron fillings in the column and were removed by running the column again with buffer (2% FBS in PBS) and collecting the eluent into a sterile tube (50ml tube). The harvested buffer-containing parasite was centrifuged at 738 x g (2000rpm) for 10 minutes and the supernatant discarded. Pellets of schizont stage infected RBCs were diluted in the ratio 1:100 in PBS. The infected RBCs were counted in a haemocytometer under a light microscope and the concentration of the infected RBCs was obtained using the formula indicated below; Number of cells/ml = n (number of cells)/4 x dilution factor x104 Number of cells/ml = n/4 x100 x 104 The schizont rich erythrocytes were then quickly subjected to three cycles of ‘Freeze- thawing’ process in liquid nitrogen at a temperature of -196oC and in a water bath at 37oC respectively to lyse up the cells to release parasite antigens into solution. The solution was then quickly centrifuged at a rate of 26564 x g for 30 minutes using a micro centrifuge. The obtained lysate containing the crude parasite antigens was aliquoted into small cryo-tubes and kept at - 80oC until ready to be used. University of Ghana http://ugspace.ug.edu.gh 67 3.2.4 Preparation of Non-Immune Sera and Antisera of Mice for Microplate ELISA The tail of each immunized BALB/c mouse was cleaned with a piece of cotton wool partially soaked in 70% ethanol. Measuring from the tip, about 3mm length of the tail was cut with a clean pair of dissecting scissors. Blood was drawn from the tail veins by applying gentle pressure from the base of the tail toward the tip (Plate 4) and a few drops of blood collected on clean parafilm. Twenty microlitres (20 µl) of blood was then fetched into an eppendorf tube containing 1000 µl of PBS and spun at 13,200 g for 5 min at 4oC to obtain the serum. The serum of each mouse was collected into a new sterilized labeled eppendorf tube and stored at -20oC until it was used for microplate ELISA 3.2.5 Coating of Microplates for ELISA Microplate ELISA was used to investigate the production of anti-Plasmodium antibodies in laboratory bred BALB/c mice. Ninety-six well (96-well) flat bottom polystyrene microtitre plates (Immulon 2HB, Part. No. 3455, Thermo Scientific, NY, USA) were coated with 50 µl/ well of Plasmodium antigen solutions (either isolated from the urine of infected individuals or extracted from cultured parasites), comprising 0.1 µg/µl protein in Dulbecco’s phosphate buffered saline (DPBS, pH 7.4; Sigma-Aldrich, Cat. No. D5773-10L). The microplates were covered with aluminium foil and incubated overnight at 4oC. University of Ghana http://ugspace.ug.edu.gh 68 3.2.6 Screening of BALB/c Mice for Anti-Plasmodium Antibodies by Indirect ELISA Microplate ELISA was conducted to test the reactivity of sera from the BALB/c mice with PAgHU or CPfAg before immunization. This experiment was carried out to determine any background reactivity in the BALB/c mice, which is required for determination of true negative and positive samples and standardization of the ELISA. Microtitre plates previously coated with cultured Plasmodium parasite or human urinary Plasmodium proteins, PAgHU or CPfAg, respectively, were retrieved from the refrigerator at 4oC. The plates were flipped empty to remove excess unbound antigen and then banged on blotting paper to remove droplets of antigen solution. The plates were rinsed briefly with DPBS washing buffer [DPBS and 0.5% (v/v) Tween 20, pH 7.4] to remove excess unbound Plasmodium antigens and then banged on blotting paper as before. Test sera were diluted serially from 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600 up to 1:3200 in PBS and 50 µl applied to duplicate wells and incubated at room temperature 25oC for 2hrs. DPBS and DPBS washing buffer were used as blanks and negative controls respectively. The plates were flipped empty and then washed two times by carefully filling the wells with DPBS washing buffer and then flipping empty after 10 min interval each. Each well of the microtitre plates was then incubated for 1hr at 25oC with 50 µl peroxidase labelled goat anti-mouse IgGAM antibody conjugate (Abcam, Cambridge, UK; Cat No. ab6006), diluted 1:2000 in DPBS Blocking buffer {DPBS and 5% w/v casein (Wako Pure Chemical Industries Ltd., Japan, Cat. No. 030-01505)}. The plates were flipped empty as before banged and washed 5 times, to remove excess unbound enzyme conjugate. The presence of bound conjugate was revealed by addition of 100 µl/well substrate solution [40 mM 2, 2-azino-bis (3-ethylbenethiazoline-6-sulphonic acid) diammonium salt (ABTS) and 0.01% (v/v) hydrogen perioxide (H2O2) in 50 mM citrate buffer, pH 4.0] to each University of Ghana http://ugspace.ug.edu.gh 69 well. The plate was shaken briefly on a plate shaker and kept covered in the dark at room temperature to allow colour development. The colourless substrate solution changed to green in wells with bound enzyme conjugate complex, which indicates positive reaction. Wells with negative reaction remained colourless. The optical densities (OD) were read at 414 nm using micro-plate reader (Thermo Labsystems Multiskan Ascent, Model No. 354). University of Ghana http://ugspace.ug.edu.gh 70 CHAPTER FOUR IDENTIFICATION OF MALARIA INFECTED AND UNINFECTED SUBJECTS IN AN ENDEMIC POPULATION Even though various efforts have been made to control malaria in developing countries, the reduction in morbidity and mortality caused by the disease is less than expected (Wogu et al., 2013). Limitations of current major diagnostic tests are a major drawback to the control efforts. For example, most of the tests are invasive, requiring the use of blood, and are not userfriendly for home-based management of malaria. Today, the application of MAbs in immunochromatographic tests to detect parasite antigens in tissue fluids, is fast gaining grounds in improved diagnostic tools for malaria, however, these tools are all invasive because they are blood-based. Even though Plasmodium antigens and anti-malaria proteins have been detected in the urine of infected individuals and are considered promising for development of diagnostic tools for malaria (Rodriguez-del Valle et al., 1991; Katzin et al., 1988; Howard et al., 2007), they are yet to be employed in a MAb-based immunochromatographic tests (ICTs) for diagnosis of the disease. In view of this, this chapter of the thesis research initiated the studies towards the development of MAbs for diagnosis of malaria, using Plasmodium parasite antigens extracted from the urine of infected individuals. In order to identify and select infected individuals from whom to obtain antigen containing urine samples for extraction of antigen and development of the malaria-diagnostic MAbs therefore, the experiments reported in this chapter were conducted firstly with the aim of identifying malaria infected individuals in the endemic population. University of Ghana http://ugspace.ug.edu.gh 71 Secondly, true negative control samples are required for ascertaining the reactivity and accuracy of the MAb-based test to be developed. To achieve this goal, therefore, this study was also conducted to identify malaria uninfected subjects in the endemic population in order to obtain true negative urine samples for ascertaining the accuracy (specificity and sensitivity) of the test and characterize its range of applicability in different geographic settings. 4.1.1 Objectives 1 Recruit study subjects and identify malaria infected and noninfected individuals by clinical diagnosis. 2 Examine blood and urine samples from study subjects to differentiate between malaria infected and non infected individuals 4.2.0 MATERIALS AND METHODS 4.2.1 Study Subject Clinical History and Baseline Characteristics The study site Kpone-on-Sea, is described in Chapter 3, section 3.1.1. The clinical history of each study participant together with age, sex and body temperature were taken before sample collection. Briefly, subjects were asked if they had taken any antimalarial chemotherapy and those who responded yes were exempted from the study. Following this, the body (axillary) temperature of subjects was taken using a digital thermometer to ascertain their febrile statuses before urine sample collection. With respect to this, the digital thermometer was placed in the ampit of each participant until temperature reading had become stable. The thermometer was then withdrawn, and the temperature reading was noted and recorded. Finally, parameters such as age and sex of participants were also obtained and duly recorded. University of Ghana http://ugspace.ug.edu.gh 72 4.2.2 Urine Sample Collection and Analysis 4.2.2.1 Urine analysis by Multistix 10 SG reagent strips for biochemical characteristics About 20-150 ml of fresh clean catch urine was collected from each study participant in the period between 08:00 and 14:00 hours Greenwich Mean Time (GMT) into a 150 ml urine container before administration of anti-malarial chemotherapy. Immediately after collection, a preliminary urine analysis was carried out on the samples using the Multistix 10 SG reagent strips (Miles Diagnostics, Elkhart, Ind.). Briefly, the urine was mixed and a urine test strip was dipped into each sample according to the manufacturer’s specification. The strip was then marched with a measuring chart on the strip container to determine the presence and amounts of malaria biomarkers, such as glucose, ketones and bilirubin. The samples were then transported to the laboratory at the Kpone Health Centre within 30 min on ice in ice-chest and then stored in a deep freezer at -20oC for further examination and analysis. The urine samples were later retrieved from the freezer, and then transported again on ice in-chest to the main laboratories at the School of Public Health and the Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, where part of each sample was aliquoted into a 14 ml Falcon centrifuge tube and then stored frozen at -20oC till used. 4.2.3 Blood samples collection and analysis 4.2.3.1 Examination of blood samples by blood smear microscopy The fourth finger of each subject was pricked with a lancet to let a drop of blood onto a glass microscope slide at each end of a glass microscope slide. The blood drop was spread into a thin film on one end, and a thick film on the other end of the slide, using the edge of a second University of Ghana http://ugspace.ug.edu.gh 73 microscope slide. The blood smear was allowed to air dry and then dipped briefly in absolute methanol to fix it onto the slide. The slide was placed in a glass staining jar containing 5% giemsa stain for 30 min and then washed gently in tap water. Each slide was air dried for at least 30 min and a drop of immersion oil was dispensed on the stained blood smear preparation to enhance visibility. The thin blood film preparation was then examined under the ordinary light microscope for parasite shape and size to ascertain parasite species. The thick blood film preparation however was examined to determine parasite density/ load. Also, in low parasite density cases, the thick blood smear was used in microscopic diagnosis to enhance sensitivity. Plasmodium parasite density was estimated as the number of parasites per unit volume of blood (e.g. 6,000 paras./μl). In this respect, parasites on a stained thick film were counted per 200 leukocytes, and then multiplied by an assumed standard count of 8000 leukocytes/μl (WHO, 2010). Figure 5 Morphological views of the various stages of the four (4) main human malaria parasite species University of Ghana http://ugspace.ug.edu.gh 74 4.2.3.2 Analysis of blood samples by micro hematocrit centrifugation Finger prick blood from each individual was also analyzed by the hematocrit system to determine hemoglobin concentration as an index of anemia as described by (Klee et al., 2000) with modification. Briefly, finger pricked blood from each subject was sampled into an anticoagulant coated (heparinized) hematocrit micro-capillary tube until it was 100 mm in height. The end of the tube that has not been contaminated by blood was then sealed with sealing wax. Each tube was inverted 10 times to allow the anticoagulant to mix with the blood sample thoroughly. The blood was then centrifuged using the The Kubota Model 3100 micro-hematocrit centrifuge (Kubota Corporation, Tokyo, Japan), at 15 000 x g for 5 min to obtain a 3 layered column of cells. The bottom opaque red layer of blood cells, representing the packed cell volume, a thin middle layer representing the buffy coat which comprised white blood cells and platelets, with the topmost transparent layer being blood plasma as represented in the Figure 6a and b below. The height of the packed red blood cell volume (PCV) was then read as a percentage of the total of 100 mm and the value obtained then divided by 3 to obtain the hemoglobin concentration of each blood sample. University of Ghana http://ugspace.ug.edu.gh 75 Figure 6: Analysis of blood by hematocrit system for haemoglobin concentration  Children: 11 to 13 gm/dL  Adult males: 14 to 18 gm/dL  Adult women: 12 to 16 gm/dL University of Ghana http://ugspace.ug.edu.gh 76 4.2.3.3 Analysis of blood samples by the quantitative buffy coat (QBC) method To ascertain the accuracy of microscopic examination (quality control), finger prick blood samples were collected from 143 patients during blood smear preparation into QBC capillary tubes pre-coated with a fluorescent dye (FD). Tubes were inverted 5 times to allow FD to mix with the blood and stain any Plasmodium parasites present. Tubes were centrifuged at 10, 000 x g for 5 min and then placed in a QBC reader to measure the level of the component hemoglobin, platelets, leukocytes, etc. Each of the tubes was finally examined under a fluorescent microscope for Plasmodium parasites, which if present, were visible as light green fluorescent grains. The Parasite-negative QBC capillary blood samples on the other hand, appeared dark under fluorescent microscope. 4.3.0 RESULTS 4.3.1 Characterization of Plasmodium falciparum infected subjects from a malaria infected population In order to select Plasmodium infected subjects to be included in the study from the total population, the clinical history and some demographic characteristics of individuals from the community were taken to ascertain their suitability for the study. Also, blood samples from these individuals from the community were examined by blood smear microscopy, Quantitative Buffy Coat (QBC) test and hematocrit centrifugation for determination of their status of infection and blood haemoglobin concentration. Furthermore, aliquots of urine samples from study subjects were examined by the BioRad protein assay for protein concentration determination as described in section 5.2.5. The data from the various experiments and measurements performed were analyzed individually and in combination to ascertain their overall impact on malaria infection outcomes and suitability of inclusion in the study. The following tables and figures therefore University of Ghana http://ugspace.ug.edu.gh 77 summarize the results of the analyses of the findings made in the various experiments conducted in this chapter. 4.3.1.1 Study Subject Clinical History and Baseline Characteristics Table 3 and Figure 7 below, summarizes the results of the analyses of the data collected from the clinical history, hematocrit centrifugation and malaria microscopy. As shown, 1262 individuals were recruited in the study. This comprised 775 (61.41%) females and 487 (38.59%) males. In all, 587/1262 (46.51%) of the participants recruited were malaria positive, of whom 321/1262 (25.44%) were females. Also, of the 587 infected population, 321 (54.68%) were females. In all, 479 individuals representing 37.96% of the total sample population had fever (Axillary temperature ≥ 37.5oC). A significantly higher proportion (358/479) of the febrile study participants, representing 74.74% or 28.37% (358/1262) were malaria positive (P = 0.0001) by microscopy, thus indicating that malaria is associated with fever among the study participants. Furthermore, 655 of the study participants representing 51.90% of the total sample population had anaemia. Out of this, 291 individuals representing 23.06% (291/1262) or 44.42% (291/655) had malaria while the simple majority (364/1262) representing 28.84% or 55.58% (364/655) were negative by microscopy. Pearson Chi-Square analysis of this results indicated that anaemia in the subjects is not associated with malaria (P = 0.173). Also, generally, relative to age, a majority, 618 (48.97%) of the study participants were older than 15 years old, while the 11-15 years age group of individuals formed the minority of 156 (12.36%). University of Ghana http://ugspace.ug.edu.gh 78 Table 3: Study Subject Clinical History and Baseline Characteristics Total Number of Individuals that have the Given Condition or Parameter Sex Total no (%) Male {No (%)} Female {No (%)} 1262 (100) No. of individuals recruited 487 (38.59) 775 (61.41) 1262 (100) Malaria positive 266 (21.08) 321 (25.44) 587 (46.51) Malaria negative 221 (17.51) 454 (35.97) 675 (53.47) Individuals with fever (Axil temp ≥ 37.5) 222 (17.59) 257 (20.36) 479 (37.96) Individuals without fever (Axil temp < 37.5) 255 (20.21) 528 (41.84) 783 (62.04) Malaria positives with fever 174 (13.79) 184 (14.58) 358 (28.37) Malaria negatives with fever 48 (3.80) 73 (5.78) 121 (9.59) Individuals with anaemia 244 (19.33) 411 (32.57) 655 (51.90) Individuals without anaemia 243 (19.26) 364 (28.84) 607 (48.09) Malaria positives with anaemia 128 (10.14) 163 (12.92) 291 (23.06) Malaria negatives with anaemia 112 (8.87) 242 (19.18) 364 (28.84) Individuals < 6 years of age 118 (9.35) 108 (8.56) 226 (17.91) Individuals from 6–10 years old 116 (9.19) 146 (11.57) 262 (20.76) Individuals from 11-15 years old 86 (6.81) 70 (5.55) 156 (12.36) Individuals >15 years of age 167 (13.23) 451 (35.74) 618 (48.97) Malaria positives <6 years old 64 (5.07) 56 (4.44) 120 (9.51) Malaria positives 6-10 years old 77 (6.10) 81 (6.42) 158 (12.52) Malaria positives 11-15 years old 55 (4.36) 33 (2.61) 88 (6.97) Malaria positives > 15 years old 70 (5.55) 151 (11.97) 221 (17.51) 4.3.1.2 Association between Malaria and Sex of Study Participants Table 4 is a summary of the result of the determination of the association between malaria and sex of study participants. According to the results, 54.7% (321) out of the 587 individuals with malaria parasite infection are females. Pearson Chi-Square analysis of the results indicated that malaria is strongly associated with sex among the study participants (P = 0.0001). University of Ghana http://ugspace.ug.edu.gh 79 Table 4: Association between Malaria and Sex Malaria Infection Rates Sex Total Female (F) Male (M) Malaria Negative Individuals (0) Count 454 221 675 % within Malaria Infection 67.3% 32.7% 100.0% % within Same Sex 58.6% 45.4% 53.5% Malaria Positive Individuals (1) Count 321 266 587 % within Malaria Infection 54.7% 45.3% 100.0% % within Same Sex 41.4% 54.6% 46.5% Total (0 + 1) Count 775 487 1262 % within Malaria Infection 61.4% 38.6% 100.0% % within Both Sex 100.0% 100.0% 100.0% P-value = 0.0001 4.3.1.3 Prevalence of Malaria among Study Subjects Relative to Age, Parasite Density and Grading Figure 7 below describes the prevalence of malaria parasite infection among study subjects in relation to age, parasite density and grade of parasitemia. According to the results, when parasite density in the subjects is ranging from low to moderate (0 to 49,000 paras/µl) and grade of parasitemia is ranging from negative (-) to 2+, subjects of age above 15 yrs had the highest prevalence of infection (i.e., 59.1%, 49.8%, and 35.6%). However, when parasite density increases beyond 50,000 paras/µl of blood, with a corresponding grade of parasitemia raging from 3+ and above in the subjects, children of ages below 6 yrs and those within the 6 to 10 yrs age group had the highest prevalence of infection. University of Ghana http://ugspace.ug.edu.gh 80 Figure 7: Prevalence of Malaria among Study Subjects Relative to Age, Parasite Density and Grading 4.3.1.4 Prevalence of Parasitaemia among Plasmodium Parasite Positive Subjects Relative of Age Table 5 below summarizes the prevalence of Plasmodium parasite load among infected study participants in relation to age. According to the results severe (50,000 – 99,999 paras./ µl) and very severe (≥ 100,000 paras./ µl) malaria are more predominant in the youngest study participants than the older counterparts. The results show that, generally, Plasmodium parasite density is very heavy among study participants of ages from below 6 to 10 years of age and reduces as they grow older from 11 years and beyond. In respect of this therefore, children within the under 6 and 6-10 years of age have the highest prevalence, 37.90 and 40.90% respectively, of 15.40% 13.60% 18.80% 34.00% 37.90% 15.30% 21.40% 28.80% 34.00% 40.90% 10.20% 15.20% 16.80% 14.00% 13.60% 59.10% 49.80% 35.60% 18.00% 7.60% 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% 70.00% No MPS 1+ [40 - 4999] 2+ [5000 - 49999] 3+ [50000 - 99999] 4+ [ >100000]P re va le n ce o f M al ar ia ( % ) Parasite Density and Grade of Parasitaemia (χ2 = 113.202; p-value=0.001) Under 6yrs 6 -10yrs 11 -15yrs Above15yrs University of Ghana http://ugspace.ug.edu.gh 81 heaviest parasite density. Furthermore, the results show that study participants of age above 15 years have the highest prevalence (49.80%) of low grade parasitaemia as compared to their counterparts within the younger age groups. In all, the results show that the prevalence of Plasmodium parasite infection among the study subjects is associated with age (P = 0.001). Table 5: Prevalence of Parasitaemia among Plasmodium Parasite Positive Subjects Relative to Age Parasite density Parasites/ul The number of individuals harbouring parasites (Parasite Prevalence) in relation to age below 6 years 6-10 years 11-15 years Above 15 years 40 – 4,999 13.60% 21.40% 15.20% 49.80% 5,000 – 49,999 18.80% 28.80% 16.80% 35.60% 50,000 – 99,999 34.00% 34.00% 14.00% 18.00% ≥ 100,000 37.90% 40.90% 13.60% 7.60% University of Ghana http://ugspace.ug.edu.gh 82 4.3.1.5 Prevalence of Malaria in Relation to Age of Study Participants The results (Figure 8), showed that the prevalence of Plasmodium parasite infection was 53.10% in children below 6 years. It then increased to the highest peak of 60.31% in the 6-10 years age group, before reducing with increasing age of participants to 56.41% among individuals within the 11-15 age category, and finally, to 35.76% in individuals of age above 15 years. The results also showed that the prevalence of the disease among participants of age from 6 to 15 years was higher than that in children below 6 years. Figure 8: Prevalence of malaria among study participants in relation to age 53.10 60.31 56.41 35.76 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Under 6 6-10 11-15 Above 15 P re v a le n c e o f M a la ri a ( % ) Age (Years) University of Ghana http://ugspace.ug.edu.gh 83 4.3.1.6 Variation in Mean Parasite Density Among Malaria Positive Subjects in Relation to Age Figure 9 below summarizes the variation in mean parasite density in malaria positive individuals in relation to age. As shown, the mean parasite load in malaria positive subjects reduces with increase in age of subjects from below 6 years to 15 yrs and above. Also, the reduction in mean parasite density among study subjects relative to age is linear. Furthermore, in conformity to the normal kinetics of malaria infection, children below 6 yrs harbor the highest mean parasite load of 52,064 paras/µl of blood whilst individuals of age above 15 yrs have the least parasite density of 9,600 paras/µl of blood. Figure 9: Variation of Mean Parasite Density Among Malaria Positive Subjects in Relation to Age 52064.28 40607.29 24524.72 9600.4 0 10000 20000 30000 40000 50000 60000 Under 6yrs 6 -10yrs 11 -15yrs Above15yrs M ea n P ar as it e D en si ty ( N o /u l) Age University of Ghana http://ugspace.ug.edu.gh 84 4.3.1.7 Association between Malaria and Anaemia Among Study Participants Table 6 below summarizes the association between the prevalence of malaria and anaemia among the study participants. The results show that 53.9% (364/675) of the malaria negative individuals have anaemia while a considerably lower percentage (49.6%) of the malaria positive subjects (291/587) than expected have anaemia. A Pearson’s Chi-Square test of significance of the relationships of these conditions shows that malaria is not associated with anaemia among the study subjects (P = 0.173). Table 6: Association Between Malaria and Anaemia Prevalence Rates Anaemia Total Negative (0) Positive (1) Malaria Negative Individuals (0) Count 311 364 675 % within Malaria Infection 46.1% 53.9% 100.0% % within Anaemia 51.2% 55.6% 53.5% Malaria Positive Individuals (1) Count 296 291 587 % within Malaria Infection 50.4% 49.6% 100.0% % within Anaemia 48.8% 44.4% 46.5% Total (0 + 1) Count 607 655 1262 % within Malaria Infection 48.1% 51.9% 100.0% % within Anaemia 100.0% 100.0% 100.0% P-value = 0.173 University of Ghana http://ugspace.ug.edu.gh 85 4.3.1.8 Variation of body temperature in different age categories of malaria infected and uninfected study subjects As shown in Figure 10, the mean body temperature of malaria positive subjects in different age groups was generally above 37.5oC. The temperatures ranged from 37.9oC in subjects below 6 years of age to 37.5 oC in subjects above 15 years of age. Fever in malaria positive individuals rose sharply from 37.9oC in children under 6 yrs age group, to the maximum value of 38.2oC in children within the 6-10 years age group before decreasing gradually to the minimum value in subjects above 15 years of age. The results as indicated in the figure, also showed that whereas all malaria positive subjects had fever (mean body temp > 37.5 oC) their malaria negative counterparts had normal body temperatures, ranging from a maximum of 37.11oC in children under 6 years to a minimum of 36.97oC in subjects above 15 years of age. Furthermore, the variation of fever among the study participants in relation to age, as shown in Figure 10, is similar to that in the prevalence of malaria among study participants in relation to age (Figure 8). A Pearson’s Chi-Square test of significance of the relationship between malaria and fever (Table 7), showed that malaria was significantly associated with fever among the study participants (P = 0.0001). University of Ghana http://ugspace.ug.edu.gh 86 Figure 10: Variation of Mean body (Axillary) Temperature of Study Subjects in Relation to Malaria Infection and Age 37.897 38.242 38.028 37.529 37.114 37.09 37.035 36.971 36 36.5 37 37.5 38 38.5 Under 6yrs 6 -10yrs 11 -15yrs Above15yrs M ea n A xi lla ry T em p er at u re /o C Age Positive Negative University of Ghana http://ugspace.ug.edu.gh 87 4.3.1.9 Association between Malaria and Fever Among Study Participants The association between malaria and fever among the study participants has been cross-tabulated in Table 7 below. Analysis of the results by Pearson’s Chi-Square test of significance indicated that malaria is significantly associated with fever (P-value = 0.0001). Table 7: Association between malaria and fever Prevalence Rates Fever Total Negative (0) Positive (1) Malaria Negative Individuals (0) Count 554 121 675 % within Malaria Infection 82.1% 17.9% 100.0% % within Fever 70.8% 25.3% 53.5% Malaria Positive Individuals (1) Count 229 358 587 % within Malaria Infection 39.0% 61.0% 100.0% % within Fever 29.2% 74.7% 46.5% Total (0 + 1) Count 783 479 1262 % within Malaria Infection 62.0% 38.0% 100.0% % within Fever 100.0% 100.0% 100.0% 4.4.0 DISCUSSION The studies conducted in this chapter aimed at characterizing the prevalence of malaria in an endemic population using Plasmodium infection outcomes with a view to identifying and selecting infected and uninfected human urine samples for generation of monoclonal antibodies towards development of rapid urine-based test for diagnosis of malaria. Prevalence of malaria among study participants In the studies described in this chapter, the prevalence of malaria was determined in the study population to be 46.51%. Even though this current prevalence was lower than that University of Ghana http://ugspace.ug.edu.gh 88 obtained in a pilot study (52.0%) conducted 9 years earlier (McKakpo et al., 2006; data unpublished) the difference was not statistically significant (P= 1.000). There was significantly (P = 0.001) higher prevalence (25.44%) of malaria among females in the study community than in their male counterparts (21.08%). A similar observation was made by other researchers like (Ayele et al., 2012), who stated that “generally, malaria parasite prevalence differed between age and gender with the highest prevalence occurring in children and females”. In spite of these observations, other research findings, have suggested that when given equal exposure, both males and females are equally susceptible to malaria infection, except for pregnant women who are more vulnerable because of the reduction in immunity that the body undergoes in order to retain pregnancy (Steketee et al., 2001). In view of this, it is worth stating that the significantly higher prevalence of malaria obtained among females within the study population could be due to factors whose determination was beyond the scope of the study. Association between malaria and age among study participants Understanding the relationship between malaria burden and age is important in selecting high-risk age groups of individuals in whom targeted intervention measures should be focused in order to avoid malaria epidemics (O’Meara et al., 2008). In line with this observation, the association of malaria with age was determined among the study population from the Kpone-on-Sea (KOS) endemic area, as part of the efforts to identify infected and uninfected urine samples for antigen extraction. Analysis of the results (Figure 7; Table 5) showed that the prevalence of malaria parasite density among the participants was significantly associated with age (P = 0.0001). According to the results, as parasite density reduced from >100,000 to 40 parasites/ µl, the University of Ghana http://ugspace.ug.edu.gh 89 prevalence of younger aged participants harbouring malaria parasites also reduced from 37.9% to 13.6% for children under 6 years age, and from 40.9 to 21.4% for children within the 6-10 years old age group. Conversely, as parasite density reduced, the percentage of older study participants harbouring parasites increased from 13.6 to 15.2% among the 11-15 year old individuals and from 7.6 to 49.8% in individuals of age above 15 years. This observation confirmed what had been reported earlier among individuals living in other endemic areas that malaria parasite infection is severest in young children, and reduces with increase in age (Doolan et al., 2009). The observation may be due to the fact that the participants from 5 months, to about 6 years of age, probably lacked adequate immune protection against malaria, which therefore, led to the high prevalence of parasitaemia among them. This trend was repeated, among participants within the 6-10 year old age group probably because, the reduction in malaria transmission following intervention programmes, prevented development of adequate immunity in these children before 10 years (Doolan et al., 2009). However, beyond 10 years of age, the participants probably started developing protective immunity against the disease. It was therefore normal for the prevalence of parasitaemia to start reducing in these individuals as they advanced in age from 11 years and beyond. The results (Figure 8), also showed that the prevalence of malaria was 53.10% in children below 6 years, increased to 60.31% in the 6-10 years age group, before finally reducing with increasing age of participants to 35.76% in individuals of age above 15 years. These findings were similar to that obtained by other authors who stated that higher risks of malaria infection were observed in children, with peak prevalence occurring among children of ages between 6 and 10 years (Ayele et al., 2012). The results confirmed what other authors (Doolan et al., 2009) have reported that in areas where intervention measures are used to University of Ghana http://ugspace.ug.edu.gh 90 reduce transmission, the development of immunity against infection is delayed, causing the peak prevalence of the disease to shift to a higher age group of individuals, until immunity is developed, when the prevalence and other infection outcomes begin to fall with increasing age of the endemic population. As reviewed earlier in the literature (Tchouassi et al., 2012), KOS experienced a considerable malaria intervention using ITN, and IPT for pregnant women just before and during the time of data collection. It is highly likely that these intervention programmes caused the peak of prevalence to shift to the 6-10 year age group. Finally, the results (Figure 7) showed that mean parasite density (MPD) among the study participants was associated with age. The MPD was highest (52,064 parasites/ µl) among the children under age 6 and decreased lineally with increasing age of participants to the lowest density of 9,600 parasites/ µl in participants of age above 15 years. These trend of age-dependent variation in parasitaemia could be because the children under 6 years of age had the lowest adaptive immunity against malaria parasite infections and therefore had the least ability to reduce parasite load (Stauffer and Fischer, 2003; Doolan et al., 2009). The older aged participants on the other hand, had probably developed a more heightened immunity against the disease which increased as they grew older and older and therefore were better able to resist the multiplication of parasites within them, hence the reducing MPD with increasing age as shown by the results. Association between malaria and anaemia among study participants The association of malaria with anaemia in the study participants was determined. As reviewed by Douglas et al. (2013), acute Plasmodium parasite infection causes the removal of parasitized and unparasitized RBCs from the blood circulation through various mechanisms which result in anaemia (Douglas et al., 2013). University of Ghana http://ugspace.ug.edu.gh 91 In respect of this, anaemia is regarded as a common manifestation of Plasmodium parasite infection. Also, the World Health Organization (WHO) and the Roll Back Malaria (RBM) partnership recommended that anaemia should be used as an additional indicator for monitoring malaria burden at the community level and interventions on the national scale (Reviewed by Santana-Morales et al., 2013). Following these reports, the association of malaria with anaemia among the study participants was determined to ascertain whether or not the prevalence of anaemia in the study community could be used to characterize malaria. According to the results (Table 3), the overall prevalence of anaemia in the study population was 51.90% (655/1262). Out of this, 291 individuals representing 23.06% (291/1262) had malaria and 364 (28.84%) were malaria negative as determined by blood smear microscopy. With the common knowledge that malaria leads to anaemia in infected individuals, it was expected that the prevalence of anaemia among the malaria positive individuals as recorded in this study would be significantly higher than that among the malaria negative counterparts, however, this was not so. A chi-square test of significance showed that there was no association between malaria and anaemia in this study (P= 0.173). Also, contrary to what was expected, a higher percentage, 52.40% (354/675) of malaria negative individuals as determined by microscopy had anaemia instead of the converse. A similar observation was made in Ethiopia, sub-Saharan Africa, where Santana- Morales et al. (2013) reviewed that a numerically higher number (50.20%) of the study participants with anaemia did not have malaria. However, recognizing the fact that anaemia is a complex condition which is not caused by only malaria, it would be explained that anaemia among the study participants would probably be multifactorial, and might have been partly due to factors such as hookworm infection and malnutrition, all of which are endemic in the study population. If this latter University of Ghana http://ugspace.ug.edu.gh 92 explanation were right, then this finding only suggested that in the absence of malaria the prevalence of anaemia in the study population could reduce by about 50%, and where there is malaria, the number of people suffering from anaemia could double (Santana-Morales et al., 2013). Indeed, although it has been recommended that anaemia could be used as an additional indicator for the presence of malaria (WHO Report, 2005) it was also advised that in order to accurately establish this, the baseline haemoglobin levels in each population should be known while taking into consideration the nutritional status as well as the infection by other parasites such as hookworms which also depletes RBCs and cause anaemia and other haemoglobin disorders in endemic populations (Santana-Morales et al., 2013). Furthermore, several studies conducted by (Steenkeste et al., 2010; Okell et al., 2009) have shown that microscopy is not sensitive enough, and often leads to underestimation of malaria prevalence.. Indeed, in a more recent study, using the improved PCR technique that enables detection of sub-microscopic Plasmodium parasite infection, Mahajan et al. (2012) reported that approximately 21% of the 101 blood samples that tested negative by blood smear microscopy, tested positive by the improved PCR-technique. It is however interesting to note that the 101 samples being referred to above, were collected from our KOS study community, prepared and examined microscopically by the same qualified technicians who assisted in the processing and analysis of the 1262 blood samples whose results are being discussed. In view of the foregoing, it would be tenable to suggest that the observation made above that there was no association between malaria and anaemia was also probably because the prevalence of malaria as determined in the sample population might have been underestimated by blood smear microscopy. The polymerase chain reaction (PCR) technique was, however, not used to ascertain the association between malaria and anaemia, because of lack of logistics. University of Ghana http://ugspace.ug.edu.gh 93 Association between malaria and fever among study participants The use of fever, that is, increase in body (axillary) temperature from 37.5oC and above, as a marker for diagnosis of malaria parasite infection forms part of the proposed recommendations of the Integrated Management of Childhood Illness (IMCI) (Reviewed by Okiro and Snow, 2010). To determine whether fever among the study participants was caused by malaria parasite infection or not, therefore, the association of malaria parasite infection with fever was estimated. Fever, pyrexia or increase in body (axillary) temperature of humans above the normal range (36.5 - 37.5oC), is said to be caused by inflammation (Laupland, 2009; Wikipedia, 2014). Inflammation is a complex innate immunological reaction mounted by the body in response to injury or tissue damage by disease causing organisms, including malaria parasites. According to the results (Figure 10 and Table 7), the prevalence of fever among the study participants was significantly associated with Plasmodium parasite infection (P = 0.0001). Also, the results (Figure 10), showed that the mean body temperature of all the malaria positive subjects across all the various age groups was greater than 37.5oC. In these malaria positive subjects, the prevalence of the disease was 61.0% (358/587). Since malaria leads to increase in body temperature, it is highly probable that fever among the study participants was due to malaria parasite infections as observed from the results. Further studies are however required to rule out the possible involvement of other concomitant infections like those caused by bacteria, viruses and measles (ECDPC Report 2013; CDC and WHO, 1998) which can also contribute to fever in the participants. Such studies were however not carried out because they were not part of the study objectives. The results (Figure 10) also showed that the variation in body temperature among the study participants with respect to age, was similar to the variation in the prevalence of malaria with respect to age (Figure 8). University of Ghana http://ugspace.ug.edu.gh 94 Since malaria was suspected as the most likely cause of fever among the study participants, variation in the age-dependent prevalence of malaria could be the only conceivable factor influencing the age-dependent severity of fever among the study participants. Therefore, the similarity of the trend in the age-dependent variation of fever to that of the age-dependent variation of malaria prevalence, is another factor to support the statement that fever among the study subjects could most probably have been caused by malaria parasite infection. Furthermore, the comparably lower and normal MBT for all malaria negative subjects (Figure 10), is once again, a factor to support the view that fever was caused by malaria parasite infection among the study participants. Since the malaria negative subjects did not have the disease, there was no expectation for fever among them. The results (Figure 10) also showed that in both malaria infected and uninfected individuals generally, the MBT was higher for children and decreased with increasing age to adulthood. This observation is similar to that reported in other literature (Antranik, 2012) where it was explained that children have higher body temperatures because of their improperly developed thermoregulatory mechanisms of the body, as well as immunity against inflammatory diseases. Therefore, as the children grow older, they develop better ability to regulate body temperature and better immunity against inflammatory infections and diseases hence, their body temperarture drops. University of Ghana http://ugspace.ug.edu.gh 95 CHAPTER FIVE EXTRACTION AND CHARACTERIZATION OF BIOMARKERS OF MALARIA FROM INFECTED HUMAN URINE 5.1.0 INTRODUCTION (1) Early diagnosis of malaria is important for prompt treatment needed to prevent complications and death, particularly, among pregnant women, children below 5 years of age and non-immune travelers (Ishengoma et al., 2011; Stauga et al., 2013). Accurate diagnosis of malaria could benefit from prior identification of specific biomarkers of the disease. Biomarkers are measurable molecular, cellular or biochemical agents or alterations in tissues or body fluids that indicate the presence and or progression of disease, or exposure to harmful substances (Kim et al., 2010; Stauga et al., 2013). Owing to their remarkable diagnostic potential, biomarkers are very useful in many aspects of health to detect infections; track the progression of disease; monitor drug delivery or metabolism; and also, to monitor drug efficacy, outcome of treatment or exposure to chemicals (Kim et al., 2010; Stauga et al., 2013). In respect of this, therefore, the development of tools for diagnosis of diseases has been largely dependent on the discovery of biomarkers of such diseases. Efforts to disvover biomarkers of diseases, have largely involved the use of blood plasma esterases (Kim et al., 2010). Also, for malaria, several biomarkers have been discovered in blood plasma and serum, which include CPR, ICAM and hemozoin (Stauga et al., 2013). However, apart from being invasive, these biomarkers may not always correlate with the severity of the disease, which undermines the sensitivity and/ or specificity of most of the tests presently used for routine diagnosis of the disease (Suaga et al., 2013). University of Ghana http://ugspace.ug.edu.gh 96 There is therefore a recognized need to expand and improve biomarker identification and quantification (Kim et al., 2010). The discovery and characterization of biomarkers of malaria, particularly, from the urine of infected individuals, is more advantageous because of several reasons. First, urine is the only biofluid that can be obtained in large quantities through non-invasive procedures (Decramer et al., 2008). Secondly, urine has been reported to contain several hundreds of biomarkers of diseases which can be explored for diagnostic purposes (Nolen et al., 2013). Also, urinary biomarkers are relatively more stable to rough handling and harsh storage conditions than other biomarkers (Nolen et al., 2013). For these reasons therefore, the studies reported in this chapter were conducted to extract and characterize biomarkers of malaria from the urine of infected individuals. 5.1.1 Aim The aim of the experiments conducted in this chapter was to isolate biomarkers of malaria from the urine of infected individuals and characterize them towards the generation of monoclonal antibodies for diagnosis of malaria. 5.1.1 (b) Specific Objectives 1) To characterize the biomarkers of malaria from infected human urine. 2) To characterize the association between urinary hepcidin, haemoglobin concentration and malaria parasitaemia. 5.2.0 MATERIALS AND METHODS 5.2.1 Characterization of the Sample Population for the Studies to Characterize Biomarkers of Malaria from Infected Urine A cohort of 290 subjects out of the larger study population participated in the pilot study reported in this chapter. They comprised 103 children and 187 adults of whom 58 were pregnant women. University of Ghana http://ugspace.ug.edu.gh 97 5.2.2 Determination of Malaria Infection Status of Study Participants Thick and thin blood smears prepared from finger prick blood and read by microscopy was used to determine the presence or absence of malaria parasites as described in section 4.2.3.1. Also, malaria parasite density (parasitaemia) among parasite positive individual was calculated as described in section 4.2.3.1. 5.2.3 Determination of Blood Hemoglobin Concentration and the Presence or Absence of Anaemia The Kubota Model 3100 micro-hematocrit centrifuge (Kubota Corporation, Tokyo, Japan) was used to determine packed cell volume of blood from which the concentration of haemoglobin in the blood of each participant was estimated as described in section 4.2.3.2 above. Also the presence or absence of anaemia was determined for each individual by comparing the haemoglobin concentration. 5.2.4 Extraction of Plasmodium Proteins from Malaria Positive Urine The isolation of Plasmodium proteins from the urine of infected individuals has been illustrated by the flow diagram below, and was carried out using the “Farr Technique” protocol described by Bosompem et al. (1996a) with modification. The Farr Technique describes the process of precipitating antigens with 33-50% saturated ammonium sulphate solution {(NH4)2SO4} (Mayer, 2010). Following aliquotting, the rest of the urine samples from positive malarial subjects, as determined by blood film microscopy, quantitative buffy coat test (QBC) and “CareStart” rapid diagnostic test for malaria, were pooled together in 3 litter conical flasks. While stirring, an equal volume of saturated ammonium sulphate solution {(NH4)2SO4} was then added to the content of the flask. The resulting solution in each conical flask was then placed in an ice- University of Ghana http://ugspace.ug.edu.gh 98 box at 4oC. The ice-box and urine solution mixture was then placed on an electronic magnetic stirrer (Stuart Scientific, United Kingdom) and then stirred till the urine-(NH4)2SO4 mixture was cloudy, an indication that urinary proteins had been precipitated out. The urinary malaria protein precipitate was then poured into 50 ml Falcon centrifuge tubes and then taken through a series of centrifugation, re-suspension with distilled water, re-addition of 50% (v/v) saturated (NH4)2SO4, stirring and re-centrifugation steps as illustrated by the flow-chart below, until the final urine pool 2J (UP2J), also called Plasmodium infected human urinary protein (PAgHU), was obtained. The final UP2J or PAgHU was then dialyzed with a cellulose dialysis tubing of membrane cuff-off pore size MWCO 12,400 (Sigma, D9527). Following dialysis, urinary Plasmodium antigen isolates were aliquotted into eppendorf micro-centrifuge tubes, examined for protein concentration using the BioRad protein assay technique (as described below) and then stored at -20oC until used. University of Ghana http://ugspace.ug.edu.gh 99 Urine Centrifuged (2,900 xg, 15min) Pellet = UP0P UP0S (Supernatant) Added; Saturated {(NH4)2SO4} 50%(V/V) Centrifuged (2,900 xg, 30min) Supernatant = UP1S Pellet Added; H2O UP1P (Suspension) Centrifuged (2,900 xg, 15min) Pellet = UP1IP UP1-IP (Supernatant) Added; Saturated {(NH4)2SO4} 50%(V/V) Centrifuged (2,900 xg, 30min) Supernatant = UP2S Pellet Added; H2O UP2P (Suspension) Centrifuged (2,900 xg, 15min) Pellet = UP2IP UP2-IP (Supernatant) Figure 11: Flow chart for extraction of Plasmodium antigens from urine of infected individuals University of Ghana http://ugspace.ug.edu.gh 100 5.2.5 Urine analysis by BioRad protein assay The aliquotted urine samples were retrieved from storage at -20oC and thawed and the pH was adjusted to between 7 and 9 to solubilize precipitates in them. Using the BioRad protein assay (Bio-Rad Laboratories 2000, CA 94547) and BSA standard reagents, the concentration of proteins in each urine sample was determined. In carrying out the experiment, bovine serum albumen (BSA) standard reagent was titrated at 1:2 dilutions (v/v) with PBS into a flat-bottomed 96-well micro-titre plate. Aliquot of each urine sample was titrated similarly, alongside the BSA reagent. Ten microlitres of BioRad protein assay reagent diluted with de-ionized water according to the ratio, three (3) parts of BioRad protein assay reagent with one (1) part (v/v) of de-ionized water, was added to each well and incubated at room temperature for 5 min with gentle shaking. The optical density of the resulting reaction in each well was measured using the micro-plate reader. A semi logarithmic graph of OD versus BSA standard reagent concentration (Cn) was plotted and then used to estimate the concentration of protein in each urine sample by extrapolation from the curve of log Cn versus OD. 5.2.6 Test Principle for Detection of Plasmodium Antigens in Samples by Different Commercial Rapid Diagnostic kits: The malaria rapid diagnostic test (RDT) cassette contains a membrane strip, which is pre-coated with mouse monoclonal antibodies (MAbs) specific to the histidine-rich protein 2 (HRP2) antigen of P. falciparum on the test line labelled P.f and/ or mouse MAbs specific to the pan-(genus) specific lactate dehydrogenase antigen (pLDH) of the four human malaria parasite species (P. falciparum, P. vivax, P. malariae and P. ovale) on the test line marked by Pan respectively. Included in the test kit is a hemolyzing buffer containing parasite (P.f or pLDH)- specific mouse MAb labelled with colloidal gold conjugate as a detectable marker. University of Ghana http://ugspace.ug.edu.gh 101 The mixture of the hemolyzing buffer-mouse MAb colloidal gold conjugate specific to HRP2 or pLDH with the Plasmodium antigen in the blood or urine sample results in the binding of the antigen to the specific antibody conjugate (figure 5). They then move along the membrane to the test regions (P.f and Pan) and form a visible line as the antibody-antigen-antibody gold particle complex which is observed as a reddish-brown line with high degree of sensitivity and specificity. Both the test line and control line in the results window are not visible before application of the sample. 5.2.7 Detection of Plasmodium Antigens in Infected Urine, Blood and Cultured P. falciparum Parasites by Rapid Diagnostic Test A rapid diagnostic test was carried out on aliquots of Plasmodium antigens (CPfAg) and (PAgHU) extracted from in vitro cultured 3D7 P. falciparum parasites and infected human urine respectively, according to the method described by Access Bio Inc (2008) with modification. Briefly, using 20-200µl ranged single channel finnipipette pipette (Thermo Fisher Scientific Incorporated, USA.), 20 µl aliquots of Plasmodium antigen extracts, and unprocessed urine samples from malaria negative and positive individuals were dispensed into the sample wells of rapid diagnostic test kit cassettes: : CareStartTM Malaria Pf/Pv combo test kit (AccessBio Inc., New Jersey, USA); SD BIOLINE malaria antigen test kit (Standard Diagnostics Inc., Republic of Korea) or First response malaria antigen Pf (HRP2) detection rapid card test (Transnational Technologies Inc., United Kingdom) for testing. Drops of buffer containing Plasmodium parasite (HRP2 or LDH) specific mouse monoclonal antibody conjugated to colloidal gold particles was then added to the buffer well of each cassette according to the manufacturer’s specification, and then allowed to develop for 15 min at room temperature (22–25 oC). Positive dipstick results showed a horizontal reddish brown line in University of Ghana http://ugspace.ug.edu.gh 102 addition to a control line on each test strip in a distinct detection zone. Negative results showed only the positive control line. During the analysis of blood, however, drops of blood were dispensed into the sample wells using the micropipettes included in the test kits according to the manufacturers’ specifications. Also, the hemolyzing washing buffer that was added was to remove the hemoglobin and allow visualization of coloured lines as shown in the diagram below. Apart from these minor differences the test on blood samples was similar to that on urine samples. 5.2.8 Detection of Malaria Proteins in Infected Urinary and Cultured Antigen Extracts by SDS-PAGE Electrophoresis of antigens was conducted using the sodium dodecyl sulfate (SDS) tris-glycine discontinuous buffer system described by Laemmli (1970). The ATTO Cooperation Slab Gel Apparatus (Bunkyo-ku, Tokyo, Japan) was used. The gel casting apparatus comprised a plastic clamp, a pair of glass slabs (one notched), a comb and U-shaped Teflon spacer. The gels were cast in a mold formed by the glass slabs assembled together and sealed with the U-shaped Teflon. The assembled apparatus stabilized by the base of the clamp holder, was mounted vertically on the working bench. A 10% separating gel (30% acrylamide mix, 1.5M Tris pH8.8, 10% SDS, 10% ammonium persulfate, TEMED, and dH20) with dimensions of 8 cm (length of separating distance) x 6 cm (width) x 0.75 mm (thickness) was cast by pouring the separating gel solution into the gel casting mold. It was overlaid gently with de-ionized water (dH2O) to prevent the surface from cracking and allowed to polymerize. The dH2O was poured off the polymerized gel and rinsed with excess de-ionized dH2O. The remainder of the gel mold was filled with 5% stacking gel solution (30% acrylamide mix, 1.0M Tris pH 6.8, 10% SDS, 10% ammonium persulfate, and dH20) after which the comb was inserted to create wells for loading of the University of Ghana http://ugspace.ug.edu.gh 103 sample preparations. The gel was then placed upright in an electrophoresis chamber filled with electrode buffer (0.25 M trizma base, 0.192 M glycine, 0.1% SDS, pH 8.3, TEMED) in both upper and lower reservoirs. The samples including extracts from Plasmodium infected and non infected urine, cultured P. falciparum antigen extracts and Plasmodium MSP119 antigen, were diluted with an equal volume of sample buffer [0.5 M trizma base (pH 6.8), 20% (v/v) glycerol, 4% (w/v) SDS, 0.02% bromophenol blue, 2.85% 2-mecaptoethanol] and then heated at 100oC for 5 min. The samples were quickly chilled after heating and kept on ice until loading. The samples were loaded (15µg/well) into the wells alongside of a molecular weight marker (mwm) and run at a constant current of 70 mA until the bromophenol blue tracking dye had entered the separating gel. The current was then reduced to 40 mA and allowed to run until the dye had almost run out of the separating gel. After electrophoresis the glass slabs were cautiously separated and the gel was left attached to one slab. The stacking gel was cut off and discarded, and a small portion of the separating gel below the tracking dye was cut off to mark the front side of the gel followed by staining or western immunoblotting. Other gels were prepared without staining using different Plasmodium infected and non-infected urine (pos and neg) and used for western immunoblotting. 5.2.9 Coomassie Brilliant Blue R-250 Staining of Gel The gel after electrophoresis, was gently placed in a container and stained with coomassie brilliant blue R-250 (CBB) solution [1% (w/v) CBB, 50% methanol, 10% Acetic acid] for 10 min with continuous rocking on an electronic shaker (Stuart Scientific-STR6). The stained gel was then de-stained in excess volume of de-staining solution-1 (40% methanol, 7% acetic acid) and then in de-staining solution-2 (5% methanol, 7% acetic acid). The gel was washed several times with excess de-staining solution 2 and then removed when University of Ghana http://ugspace.ug.edu.gh 104 the background was clear and the protein bands visible (Appendix 1). The molecular weight (Mwt) of each protein band was then determined by comparison of its relative mobility (Rm) with those of several marker proteins of known molecular weight (Mwt). A semi logarithmic graph of the logarithms of Mwt of the protein standards as functions of the Rm values was plotted and the unknown Mwt of the samples was estimated using extrapolation from the curves of log Mwt versus Rm. 5.2.10 Identification of Biomarkers of Malaria by Western blotting Analysis The gels were prepared as described above without staining. The protein bands in each gel were transferred onto PVDF membrane (Immobilon-P transfer membrane, Millipore Cooperation, Cat. No. IPVH00010) by electro-blotting using a transfer apparatus (Milliblot- Graphite Electroblotter 1, Millipore Cooperation), which had been previously immersed in transfer buffers. The PVDF membrane containing the transferred bands was then probed with different anti-Plasmodium monoclonal antibodies (MAbs) to identify the Plasmodium antigen in each band. In carrying out the experiment, two pieces of blotting paper (9 x 7 cm) were placed on the anode pole and wetted with anode buffer I (0.3 M trizma base, 10% methanol, pH 10.4). Another blotting paper was wetted with anode buffer II (25 mM trizma base, 10% methanol, pH 10.4) and placed on them. The PVDF membrane pre-wetted in methanol to remove traces of air bubbles and render it hydrophilic, was rinsed in de-ionized water (dH2O) and laid on the set of the anode buffer-wetted blotting paper. The gel containing the transferred protein bands was immediately placed on the PVDF membrane with face down. Two pieces of blotting paper wetted in cathode buffer (25 mM Trizma base, 192 mM glycine, 20% methanol, pH 9.4) was then placed carefully on the gel. A pipette was gently rolled over the blotting paper-PVDF membrane-gel-blotting paper layer to remove all traces of air University of Ghana http://ugspace.ug.edu.gh 105 bubbles. The setup was then supplied with constant current of 200 mA for 55 min after covering with the cathode pole. The PVDF membrane with the blotted proteins was rinsed in phosphate buffered saline (PBS) washing buffer and then incubated in blocking buffer [5% (w/v) skimmed milk in washing buffer] at 25oC for 30 min. The membrane was transferred into a 1:50 dilution of anti-Plasmodium falciparum histidine rich protein 2 (pfHRP2) MAb and incubated at 25oC for 2 h. The membrane was removed, rinsed once with washing buffer and then washed two times with washing buffer at 10 min intervals on a shaker at room temperature. The membrane was probed with horseradish peroxidase conjugated to goat anti-mouse IgM (Sigma, A8786) antibodies diluted 1:500 in blocking buffer. The membrane was incubated in substrate solution (100 mM phosphate buffer, pH 6.4) until blue-black bands were observed. The experiment was repeated several times with a different probing anti-Plasmodium antibody for each blot. 5.2.11 Characterization of urinary hepcidin from Plasmodium infected human urine Urinary hepcidin was measured using the surface-enhanced laser desorption/ ionization time-of-flight mass spectrometry (SELDI-TOF MS) as described by Kemna et al. (2005). Urine samples were thawed, vortexed, and centrifuged at 12,000 rpm for 25 minutes. Seven microlitres (7 uL) of the supernatant of each ample was applied in triplicate (3 spots) on an NP20 Ciphergen ProteinChip® (CPC) and incubated in a moist chamber for 30 min. Each CPC was washed three times with high performance liquid chromatography (HPLC) grade water for 10 min to remove excess/ unbound proteins. The CPC arrays were air-dried, and 1.0 µL of alpha-cyano-4-hydroxy-cinnamic acid (CHCA, Ciphergen) in 50% acetonitrile and 0.25% trifluoroacetic acid was added to the array surface and allowed to dry in air. University of Ghana http://ugspace.ug.edu.gh 106 The arrays were then analyzed on a CPC Reader (Model PBS II, Ciphergen). The data were collected using the settings described by WHO (1968) as follows. Two warming shots at laser intensity 175 (not collected); 10 shots at laser intensity 170 at every five positions between 20 and 80; high mass 4500 Da; mass deflector 100 Da, detector sensitivity 7; acquired mass range from a mass-to-charge (m/z) ratio of 500 to 4500. Peak annotation was conducted using CPC Software (version 3.2.0) after baseline subtraction and adjustment had been carried out. The peak for hepcidin was recorded at a characteristic mass-to-volume (m/v) ratio of 2790 when the signal-to-noise ratio (s/n) is > 3:1. Although the molecular weight of hepcidin is 2789, it is ionized as a charged (protonated) molecule on the SELDI-TOF mass spectrometer; thus, the average peak on low-resolution mass spectrometry is one unit higher than the average molecular weight because of the proton gained. Urinary creatinine was measured using a commercial ELISA (Quidel Corporation, San Diego, CA) according to the manufacturer’s protocol. Between run coefficients of variation (CV) for urinary creatinine were 11.9% and 5.9% for high and low controls, respectively. Urinary hepcidin concentrations were expressed as intensity per mmol/L creatinine. Means and standard deviations were calculated for descriptive statistics. Anemia was defined as hemoglobin < 11 g/dL for children and pregnant women, < 12 g/dL for non- pregnant adult women, and < 13 g/dL for men (WHO, 1968). Both malaria parasitemia and urinary hepcidin (mmol/L creatinine) were continuous variables that were highly skewed and were transformed by loge to normalize the distribution. Linear regression models were used to examine the relationship between urinary hepcidin and other variables such as parasitemia and hemoglobin. University of Ghana http://ugspace.ug.edu.gh 107 5.3.0 RESULTS 5.3.1 Detection of P. falciparum HRP2 Antigen in Infected Human Urine by Commercial Diagnostic Kits Plate 1 below illustrated the results of an experiment to identify Plasmodium antigens in infected human urinary antigen extract (PAgHU). According to the results, the anti-P. falciparum histidine-rich protein 2 (HRP2) antibody-based rapid diagnostic test (RDT) kit was reactive to PAgHU as shown by the test result line labelled “T” in the plate below. The mark labelled “C” was the test control line, which indicated that the test was successful. Positive Control line P. falciparum HRP2 antigen-positive band Plate 1: A commercial RDT cassette showing the presence of P. falciparum histidine rich protein II (pfHRP2) in crude antigens (PAgHU) extracted from malaria infected human urine. C, is the control line, Pf is the pfHRP2 band. University of Ghana http://ugspace.ug.edu.gh 108 5.3.2 Detection of Plasmodium LDH Antigens in Infected Human Urine by the CareStartTM Rapid Diagnostic Kit Plate 2 below illustrated the results of an experiment to identify Plasmodium antigens in infected human urinary antigen extract (PAgHU). According to the results, the anti- Plasmodium pan (genus)-specific lactate dehydrogenase (LDH) antibody-based RDT kit was reactive to PAgHU, as indicated by the test result line labelled “Pan” in the plate below. The mark labelled “C” was the test control line, which indicated that the test was successful. Positive control line Pan-specific Plasmodium Parasite lactate dehydrogenase Antigen (LDH) P. falciparum HRP2 antigen-positive band Plate 2: A commercial RDT cassette showing the presence of P. falciparum histidine rich protein 2 (pfHRP2) in crude antigens (PAgHU) extracted from malaria infected human urine 5.3.3 Urine analysis by BioRad protein assay Following the extraction of Plasmodium proteins from infected human urine and in vitro cultured Plasmodium parasites, the BioRad protein assay was used to determine the concentration of proteins in the antigen extracts by extrapolating sample ODs on a standard University of Ghana http://ugspace.ug.edu.gh 109 curve of bovine serum albumen (BSA) (Fig 32, below) and the results show that the concentration of proteins in the human urinary antigen extract (PAgHU) was 0.75 mg/ml (0.75g/l) whilst that of the in vitro cultured P.falciparum pararasite antigen extract (CPfAg) was 0.50 mg/ml. Figure 12: Determination of Concentration of Proteins in Plasmodium Infected Urine by BioRad Assay 0.0000 0.2000 0.4000 0.6000 0.8000 1.0000 1.2000 0.78 1.56 3.13 6.25 12.50 25.00 50.00 100.00 O p ti ca l D e n si ty O D 6 3 0 BSA Prontein Concentration (mg/ml) x 10-2 Standard Protein (BSA) Assay Curve OD1 (nm) Linear (OD1 (nm)) University of Ghana http://ugspace.ug.edu.gh 110 5.3.4 Characterization of proteins in Different Urine and Plasmodium Antigens by SDS-PAGE Figure 13 below, summarized the results of the SDS-PAGE experiment to characterize the proteins within different antigen preparations. As shown, PAgHU, lane 2, was one of the 2 proteins with the highest number (10) of bands within the protein profile. Three (3) of these bands were most prominent and had MWs of 10.4, 27.2 and 50 kDa. The urine sample from the 3+ malaria positive individual (DUMI-2, lane 8), was the other antigen preparation with the highest number (10) of proteins within the profile. Two of its most prominent bands had MW of 27.2 and 63 kDa. The 2+ malaria infected urine sample (DUMI-3, lane 9), had the profile with the next highest number of bands. The profile contained 6 bands, 2 of which had joined together to form a thick prominent band. The third band which was also prominent and located slightly above the merged bands, had a MW of 27.5 kDa. DUMI-3 had another prominent band with MW being 63 kDa like DUMI-2, but which was less prominent than those described earlier. A urine sample from 1+ malaria positive individual (DUMI-4, lane 10) had the least number (3) of visible bands of MW, 10.4, 27.5 and 32 kDa, of which the 10.4 kDa protein was the most prominent. In addition to the antigen samples described above with bands in their profile, was the Plasmodium MSPl19 antigen (lane 6) which had a huge prominent band located below the 7 kDa MW level. Finally, the remaining 3 samples, comprising the malaria negative urine from Ghana (lane 3), malaria negative urine from USA (lane 4), and the cultured Plasmodium parasite antigen extract (CPfAg, lane 5) did not have any observable bands within their profiles. University of Ghana http://ugspace.ug.edu.gh 111 1 2 3 4 5 6 7 8 9 10 Figure 13: Coommassie blue gel electrophoresis results Lane 1 Prestained SDS-PAGE broad range molecular weight marker Lane 2 Crude urinary Plasmodium antigen extract (PAgHU) Lane 3 Urine from Plasmodium negative individual from Ghana Lane 4 Cultured 3D7 strain P. falciparum parasite antigen extract Lane 5 Urine from Plasmodium negative individual from USA Lane 6 Plasmodium MSP119 antigen Lane 7 Plasmodium parasite aldolase antigen Lane.8 Urine from 3+ Plasmodium positive individual Lane 9 Urine from 2+ Plasmodium positive individual Lane 10 Urine from 1+ Plasmodium positive individual Protein Molecular Weight 175,000 80,000 58,000 46,000 30,000 25,000 17,000 7,000 University of Ghana http://ugspace.ug.edu.gh 112 1 2 3 4 5 6 8 λ δ π 5.3.5 Plasmodium Proteins in Different Antigen Preparations by Western Blotting Analysis Figure 14 showed the results of the western blotting experiment to identify Plasmodium proteins in 6 antigen preparations using mouse anti-Plasmodium polyclonal antibodies (PAbs). As shown, the mouse anti-Plasmodium PAb detected bands in protein profiles of only 2 of the 6 antigen preparations analyzed. These antigen preparations were the PAgHU and the direct urine sample from a 3+ malaria positive individual (DUMI-2). According to the results, the PAb detected fewer protein bands in the profile of PAgHU than that of DUMI-2. The 2 protein bands detected in PAgHU comprised a 50.9 kDa antigen labelled Ω and a 54.6 kDa antigen labeled β. Out of these 2 proteins, the band size of Ω was bigger and darker compared to that of β. On the other hand, the 3 proteins detected by PAb within the profile of DUMI-2 were a 27.2, 46 and a 52.7 kDa proteins labelled π, δ and λ respectively. Also, these results showed that the band size of the protein labelled λ was larger and darker than those of δ and π. Figure 14: Western blotting results Lane 1 Prestained SDS-PAGE broad range molecular weight marker Lane 2 Crude urinary Plasmodium antigen extract (PAgHU) Protein Molecular Weight 175,000 80,000 58,000 β Ω 46,000 30,000 25,000 17,000 7,000 University of Ghana http://ugspace.ug.edu.gh 113 Lane 3 Urine from Plasmodium negative individual from Ghana Lane 4 Urine from Plasmodium negative individual from USA Lane 5 Cultured P. falciparum crude antigen extract (CPfAg) Lane 6 Plasmodium MSP119 antigen Lane 8 Urine from 3+ Plasmodium positive individual 5.3.6 Characterization of Hepcidin from Plasmodium Infected Human Urine Table 8 summarized the results of the experiments to characterize hepcidin as a biomarker for malaria in the urine of 199 malaria infected and uninfected subjects whose infection status had been determined earlier by blood smear microscopy. As shown, out of the 199 study participants whose urine samples were examined, 83 (41.7%) were children, 82 (41.2%) were adults and 31 (15.6%) were pregnant women. Urine samples from 3 (1.5%) of the participants who were children, were not included in the study because of the lack of data on their ages. This therefore made the total number of children involved in this aspect of the study to be 86 (43.2%). According to the results, the prevalence of malaria among this cohort of study population was 69.1% among children, 44.7% among adults and 41.4% among pregnant women. Also, the prevalence of anaemia among this study participants was 79.1% among children, 82.9% among adults and 90.3% among pregnant women. In addition, the results showed that the minimum and maximum levels of parasitaemia among these study participants were 40 and 394,480 among children, 40 and 195,040 among adults and 40 and 8,960 among pregnant women. Furthermore, the results showed that the minimum and maximum haemoglobin levels among the study participants were 3.8 and 12.6 g/dL among children, 7.9 and 17.3 g/dL for adults and 6.2 and 12.6 g/dL for pregnant women. Finally, the mean urinary hepcidin concentration among healthy control individuals, as determined by the experiments, was 0.52 intensity/ mmol creatinine, while that among the University of Ghana http://ugspace.ug.edu.gh 114 infected individuals ranged from 3-6 intensity/ mmol creatinine. These results were however not shown in the data summarized below. Table 8: Characterization of Hepcidin from Plasmodium Infected Human Urine and Study Subjects Characteristics Characteristics Mean (SD) or %, unless noted Children (n = 83) Adults (n = 82) Pregnant women (n = 31) Age 7.9 (5.1) 31.3 (13.0) 24.4 (5.1) Female (%) 44.2 56.1 N/A Haemoglobin concentration (g/dL) 9.5 (1.8) 11.3 (1.6) 9.0 (1.7) Anaemia (%) 79.1 82.9 90.3 Log parasitaemia 6.84 (4.36) 3.45 (3.98) 3.95 (3.56) Geometric mean parasitaemia (parasites/ 200 white blood cells) 934 31.5 51.9 Hepcidin concentration (intensity/ mmol creatinine) 4.2 (1.4, 9.5) 0.7 (0.3, 3.6) 0.7 (0.2, 5.9) 5.3.7 Association between urinary hepcidin level and parasitaemia, anaemia and haemoglobin level Table 9 summarized the relationship between urinary hepcidin levels and parasitaemia, haemoglobin and anaemia in each of the 3 study groups, and in all the 3 groups combined, as determined by uni- and multi-variable linear regression models. According to the results, examination of the data by a univariable linear regression model indicated that the level of hepcidin (intensity/ mmol creatinine) in subjects urine was significantly associated with log parasitaemia among the different study participant groups comprising 86 children (regression coefficient, β = 0.086, standard error, SE = 0.035, P < 0.017); 82 adults (β = 0.184, SE = 0.043, University of Ghana http://ugspace.ug.edu.gh 115 P < 0.0001); 31 pregnant women (β = 0.218, SE = 0.085, P < 0.016) and in all groups combined (β = 0.171, SE = 0.025 and P < 0.0001). On the other hand, urinary hepcidin levels were not significantly associated with haemoglobin levels in the children (P = 0.58), adults (P= 0.79), pregnant women (P = 0.54) and in all the groups combined (P = 0.10). Also, urinary hepcidin levels were not significantly associated with anaemia in the children (P = 0.38), adults (P = 0.07), pregnant women (P = 0.54) and all the groups combined (P = 0.13). Furthermore, examination of the data by a multi-variable linear regression model showed that loge urinary hepcidin was significantly associated with loge parasitaemia among the study participants (β = 0.174, SE = 0.026, P < 0.0001), but not loge haemoglobin levels (β = 0.022, SE = 0.058, P = 0.71). In addition, the results showed that there was no significant association between loge parasitaemia and haemoglobin levels as determined by the linear regression models. Finally, examination of the severely anaemic individuals data by an alternative multivariable linear regression model also showed that loge urinary hepcidin was significantly associated with loge parasitaemia (β = 0.248, SE = 0.056, P < 0.0001), but not haemoglobin levels (β = 0.211, SE= 0.192, P = 0.28). University of Ghana http://ugspace.ug.edu.gh 116 Table 9: Association between urinary hepcidin level and parasitaemia, haemoglobin level and anaemia Characteristics Regression coefficient Beta 95% Confidence interval (CI) Standard error (SE) P-value (P) Children (n = 83) Log parasitemia 0.086 0.016-0.156 0.035 0.017 Anemia 0.048 −0.123–0.221 0.086 0.58 Hemoglobin 0.345 −0.426–1.112 0.388 0.38 Adults (n = 82) Log parasitemia 0.184 0.099–0.268 0.043 < 0.0001 Hemoglobin −0.030 −0.262–0.201 0.116 0.79 Anemia 0.900 −0.068–1.869 0.487 0.07 Pregnant women (n = 31) Log parasitemia 0.218 0.043–0.392 0.085 0.016 Hemoglobin −0.110 −0.516–0.276 0.193 0.54 Anemia 0.677 −1.541–2.986 1.083 0.54 All groups combined (n = 196) Log parasitemia 0.171 0.122–0.221 0.025 < 0.0001 Hemoglobin −0.101 −0.222–0.020 0.061 0.10 Anemia 0.481 −0.143–1.106 0.317 0.13 University of Ghana http://ugspace.ug.edu.gh 117 5.4.0 DISCUSSION The studies described in this chapter sought to characterize possible biomarkers of malaria from the urine of infected individuals. The strategies adopted were partly to determine the levels of various biomarkers of malaria in the urine of infected and uninfected individuals, using the Multistix 10 SG reagent strips (Miles Diagnostics, Elkhart, Ind.), extract proteins from the urine samples and then characterize them by SDS-PAGE, western blotting assay and mass spectrometry sequencing. Finally, the strategies adopted were also to determine the level of association of the identified biomarkers with malaria among the study participants. Identification of biomarkers of malaria in urine samples by commercial rapid diagnostic test (RDT) kits The positive reactivity of the histidine rich protein 2 (HRP2) based test kit with the malaria antigen extract from infected human urine (PAgHU) suggested that probably, PAgHU contained P. falciparumi HRP2. On the other hand, the non reactivity of the test kit with the negative control urine samples suggested that the HRP2 antigen was not present in those urine samples from malaria negative individuals. The detection of HRP2 in the urinary antigen extract from malaria positive individuals, unlike the negative control urine samples, therefore, indicated that probably, an HRP2-based RDT could also be developed for diagnosis of malaria in the urine of infected individuals. Since diagnosis of malaria like other diseases is gradually shifting focus from the use of invasive methods to non-invasive techniques, this test when developed, would have an advantage over existing ones because, it is urine-based and therefore non-invasive. Also, the detection of the Plasmodium pan (genus)-specific lactate dehydrogenase (LDH) antigen in PAgHU, indicated that probably, the Plasmodium genus-specific LDH antigen was present in the urine of infected individuals. The non-reactivity with the negative control urine University of Ghana http://ugspace.ug.edu.gh 118 samples, on the other hand suggested that the antigen was not present in uninfected human urine, just like the HRP2 antigen. These results therefore implied that a urine-based commercial RDT kit could also be developed for malaria, which when available, would be more attractive to health care givers because of its non-invasive nature. Characterization of biomarkers in different antigen preparations by SDS-PAGE The experiments to characterize proteins in different fractions of urine and Plasmodium antigens by SDS-PAGE revealed that PAgHU and the direct urine samples from malaria infected individuals (DUMIs) contained higher numbers of proteins in their profiles than the negative control and the rest of the samples. It has been reported that humans infected with Plasmodium falciparum frequently have elevated levels of proteins in their urine (Rodriguez-del Valle et al., 1991). In view of this, the higher number of proteins as observed in PAgHU and DUMIs compared to the negative samples, confirmed the report that infection by malaria parasites leads to the excretion of proteins in the urine of affected individuals. The absence of such protein bands from the profiles of the negative control urine samples therefore, gave the indication that the identified proteins in the urine of infected individuals might be malaria-related biomarkers, which could be explored by further studies to determine their functions in the Plasmodium parasite, their possible association with malaria and suitability for development of non-invasive urine-based diagnostic tests for malaria. In order to obtain PAgHU, urine samples from over 400 malaria infected individuals, some of whom harboured >100,000 parasites/ ul blood, were pooled together before extraction of the antigen. Therefore, the higher numbers (>10) of protein bands in PAgHU and the 3+ malaria positive individuals’ urine (DUMI-2), which decreased with decreasing parasite density (parasitaemia) to 6 protein bands in the 2+ malaria positive individuals urine (DUMI-3) and lastly, to 3 bands in the 1+ malaria positive individuals urine (DUMI-4), suggested that the University of Ghana http://ugspace.ug.edu.gh 119 separation and identification of proteins in urine, using SDS-PAGE, could be explored to quantify parasite density in the urine of malaria infected individuals. Also, the results suggested that the separation and characterization of proteins in infected urine could be used to predict the severity of the disease in infected individuals. In addition, the fact that some of the protein bands were more prominent than others suggested that probably, the proteins with the darker bands had higher relative abundance (or concentrations) within the respective antigen preparations. The presence of only one band in the protein profile of the MSP119 antigen as shown by the results, also confirmed the fact that the MSP119 antigen preparation was a pure recombinant antigen comprising only one protein. Finally, the fact that the negative control urinary antigen preparations did not show any protein bands in their profiles suggested that probably, the donors were not suffering from any disease which could lead to the excretion of proteins in the urine. These results therefore confirms the findings that the presence of proteins in the urine was suggestive of a defective urine filtration system in the Bowman’s capsule. This condition is said to arise as a result of an infection that causes inflammation and its consequent alteration of the fine structure of the filtration membrane within the Bowman’s capsule. Characterization of biomarkers in different antigen preparations by western blotting assay In the western blotting experiment to characterize the proteins in different malaria infected urinary and Plasmodium antigen preparations, the mouse anti-Plasmodium polyclonal antibody (PAb) was observed to have reacted to five (5) protein bands within PAgHU and DUMI-2. These results suggested that probably, those protein bands were biomarkers of malaria, and should therefore be studied further for possible exploration for development of non-invasive urine-based diagnostic tools for malaria. University of Ghana http://ugspace.ug.edu.gh 120 Also, the fact that the 54.6 kDa protein labelled Ω in the profile of PAgHU and the 52.7 kDa protein labelled λ in the DUMI-2 profile had stronger reactivity to the PAb than the other protein bands in the 2 profiles, suggested that probably, those proteins had higher relative abundance in the respective antigen preparations than the others. On the other hand, it could be because, those proteins were more immunogenic, and therefore, induced the production of higher titres of antibody against those proteins than the others which reacted weakly to the PAb. Furthermore, the similarity of the intensity of the reactivity of the 2 proteins (Ω and λ) to PAb coupled with the closeness of their molecular weights also suggested that probably, the 2 proteins were isoforms of the same biomarker of malaria which were being expressed at slightly different molecular weights from each other. On the other hand, it was also possible that the method used in extracting PAgHU, might have modified the protein in PAgHU compared to DUMI-2, which was a direct unprocessed urine sample. This probable modification of the proteins in PAgHU might have led to the slight difference in molecular weight of the same protein in the 2 different antigen preparations. In addition, the fact that there was no observable reactivity between the PAb and the cultured Plasmodium crude antigen extract (CPfAg), suggested that probably, the concentration of proteins in CPfAg was far below the threshold that was necessary for identification by PAb. Indeed, the BioRad protein assay showed that the cultured parasite antigen (CPfAg) had lower concentration of proteins (0.5 µg/µL) than PAgHU which had 0.75 µg/µL of proteins. This observation therefore was a factor that supports the statement that CPfAg probably had lower than the amount of proteins required to show observable reactivity between it and the PAb. Lastly, the non reactivity of the negative control urine samples with the PAb suggested that possibly, they did not contain any malaria-related biomarker at all. University of Ghana http://ugspace.ug.edu.gh 121 Characterization of urinary hepcidin as a biomarker of malaria in the urine of study subjects: Two hundred and ninety (290) urine samples were originally obtained for identification and characterization of malaria-related biomarkers in a pilot study to ascertain the suitability of urine samples for development of a non-invasive diagnostic test for malaria. However, only 199 urine samples were finally utilized in the study that led to the discovery of urinary hepcidin as a biomarker for malaria. This was because, following the repetitive use of the urine samples for preliminary experiments, 91 urine samples ran out. Also, though 199 samples were analysed for urinary hepcidin, data from 196 samples were used in the statistical analysis, because 3 samples did not have data on the ages of the participants and were therefore excluded from the analysis. According to these preliminary studies, the highest prevalence of malaria (69.1%) recorded for children, compared to the 44.7% for adults and 41.4% for pregnant women confirmed the findings that children, especially, those below age 5, are among the group at highest risk of infection by the disease. Also, the lowest malaria prevalence rate of 41.4% that was obtained for the pregnant women among the 3 groups, was attributable to the fact that those women were part of the Intermittent Preventive Treatment (IPT) programme that pregnant women in the study community were involved in. The results showed again that contrary to expected findings, the prevalence of anaemia, unlike malaria infection, was lowest (79.1%) among children than in adults (82.9%) and pregnant women (90.3%). These results therefore suggested that probably, anaemia among the children was not attributable to malaria infection. However, the highest prevalence of anaemia obtained among pregnant women in this study confirmed reports that pregnancy induces anaemia among affected women (Anderson, 2002). University of Ghana http://ugspace.ug.edu.gh 122 Finally, the highest range of parasitaemia (40 and 394,480) among children as compared to adults and pregnant women, further supported the highest malaria prevalence rate in suggesting that children were among the most affected group in susceptibility to malaria. In addition, the lowest range of parasitaemia (40 and 8,960) that was obtained for pregnant women as compared to the children and adults supported the fact that probably, IPT contributed to the reduction of malaria infection rates among these women. Hepcidin is a recently discovered peptide hormone synthesized by liver cells, and is considered as an important modulator of iron metabolism in homeostasis. Hepcidin produced by chronic inflammation is known to block the release of iron from liver cells, macrophages and intestinal epithelial cells, leading to hypoferremia and therefore, is thought to contribute to anaemia in affected individuals. Malaria causes inflammation, and chronic inflammation leads to the synthesis of hepcidin (Anderson et al., 2002). Since both malaria and increased hepcidin concentration in the body are known to lead to anaemia, they were considered to have something in common. In view of this, in the efforts to identify and characterize biomarkers of malaria from the urine of infected individuals, the relationship between urinary hepcidin concentration and malaria parasitaemia, anaemia or blood haemoglobin levels was determined. The results showed that the mean hepcidin concentration among healthy control individuals was 0.52 intensity/ mmol creatinine while that among infected individuals ranged from 3-6 intensity/ mmol creatinine. Also, the examination of the data by the linear regression models indicated that high levels of parasitaemia was significantly associated with increased urinary hepcidin concentration. These results together suggested that increased levels of urinary hepcidin among infected individuals might have been caused by inflammation resulting from increasing malaria parasite load among the participants. The results therefore implied that urinary hepcidin concentration University of Ghana http://ugspace.ug.edu.gh 123 could be used as a biomarker for malaria parasitaemia levels. Further studies are however required to explore this finding in development of a urine-based quantitative test for malaria using hepcidin levels as a marker for diagnosis. Furthermore, the results suggested that urinary hepcidin concentration could not be used as a marker for determination of the haemoglobin or anaemia status of infected individuals. The results therefore suggested that haemoglobin levels and anaemia among individuals may be contributed by several factors, including nutrition as indicated by earlier findings (Santana- Morales et al., 2013). This study as a whole, provided the initial information on the relationship between urinary hepcidin concentration and parasitaemia among malaria infected individuals, even though it was limited by the lack of indepth studies to clarify the apparent lack of association between urinary hepcidin levels and haemoglobin and anaemia. Since these findings would contribute towards identification, characterization and development of new biomarkers for non-invasive diagnosis of malaria and its consequences, further studies are therefore required to examine the longitudinal variation in urinary hepcidin concentration and its association with changes in haemoglobin concentration, anaemic status and iron status indicators. University of Ghana http://ugspace.ug.edu.gh 124 CHAPTER SIX GENERATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST PLASMODIUM FALCIPARUM ANTIGENS USING URINARY AND IN VITRO CULTURED PARASITES 6.1.0 INTRODUCTION Global eradication of malaria has not achieved expected success (Shiff, 2002) partly because of the limitations in diagnostic accuracy (Eibach et al., 2013; Reyburn et al., 2007). Tools presently available on the market for malaria detection are invasive and sometimes involve the use of complex techniques or sophisticated equipment which are difficult to find outside the health facility (Azikiwe et al., 2012; Eibach et al., 2013). This need for blood makes most of the available tests non user friendly. To overcome these diagnostic limitations, this study seeks to explore the development of a rapid urine-based dipstick test (RUBDA) for diagnosis of the disease. For the RUBDA to function as an effective diagnostic tool, monoclonal antibodies (MAbs) may be used as essential reagents that can specifically recognize the malaria parasite antigens in diagnosis. Several authors have generated MAbs against Plasmodium antigens (Dzakah et al., 2013; Seth et al., 2013 Kattenberg, et al., 2012) some of which have been utilized in simple ICT based diagnostic tests for malaria, such as CareStart MalariaTM, Paracheck, Malaria Rapid Test, ICT Malaria and Parasight F test for detection of P. falciparum histidine rich protein 2 (Eibach et al., 2013; Reyburn et al., 2007). However, MAbs to malaria specific urinary antigens are yet to be reported. University of Ghana http://ugspace.ug.edu.gh 125 In view of this, the studies described in this chapter were conducted to generate MAbs against urinary Plasmodium parasite antigens suitable for development of the RUBDA for diagnosis of malaria using suspected patient urine. Hybridoma products from cell fusion require cloning, expansion by cell culture, harvest, purification and formulation into a target antigen -specific MAb reagent. MAbs generated through cell fusion was therefore characterized to determine antibody isotype and binding characteristics in different assay systems including micro-plate ELISA and Western Blotting analysys. 6.1.1.0 Aim The aim of the studies conducted in this chapter was to generate MAbs by cell fusion against Plasmodium antigens present in infected human urine and characterize them by isotyping and reactivity in microplate ELISA. 6.2.0 MATERIALS AND METHODS 6.2.1 Preparation of Immunogens for Immunization of BALB/c Mice The immunogen (antigen preparation) for immunization of one mouse was prepared by dispensing 15µg of antigen extract and 50µl of Freund’s complete adjuvant (FCA) into a clean 5ml glass vial, and equilibrating the volume to 100µl by addition PBS (pH 7.4). The content of the vial was thoroughly emulsified by mixing with a 2ml syringe fitted with 18xG hypodermic needle. To make provision against wastages an additional 30µl of immunogen per mouse was added to the original 100µl computed for each mouse. Also, to minimize the loss of immunogen during preparation, the antigen preparation for immunization of 10 mice was mixed together in University of Ghana http://ugspace.ug.edu.gh 126 the same vial. After preparation the immunogen was dispensed into a 1.0ml hypodermic syringe fitted with a 26G x ½ needle before inoculation into the mice. 6.2.2 Immunization of BALB/c Mice Eight to twelve weeks old BALB/c mice were immunized intraperitoneally (Plate 4) for production of anti-Plasmodium falciparum antibodies and were given three boosters at two-week intervals. A final booster a few days prior to cell fusion was also administered. Prior to immunization, 20 µl of tail vein blood was collected (Plate 3) into eppendorf tubes containing 1000 µl of PBS as shown in Plate 2. This was important for monitoring the progress of immune response in the mice. Immunization was performed by administering 15 µg/100 µl of In vitro cultured Plasmodium parasite antigen extract (CPfAg) emulsified in 50% (v/v) Freund’s Complete Adjuvant (Sigma F-5881) using 26G x ½ needles as shown in Plate 3. Booster immunization consisting of similar concentration of the same immunogen in Freund’s Incomplete Adjuvant (Sigma F-5506) was given on days 14, 28 and 42 after the first immunization. The final booster consisting of 15µg/100 µl of proteins isolated from Plasmodium infected human urine (PAgHU) without adjuvant was administered 4 days prior to cell fusion. PAgHU was used as a final booster inoculum to increase the population of B-lymphocytes bearing the gene coding for biosynthesis and secretion of anti- PAgHU antibody. The mice were bled from the tail veins before each immunization and the sera tested for the presence of anti- PAgHU antibodies using the micro-plate ELISA method. University of Ghana http://ugspace.ug.edu.gh 127 Plate 3: Collecting blood from the tail of BALB/c mouse before and after immunization Plate 4: Inoculating BALB/c mouse with PAgHU or CPfAg Immunogen University of Ghana http://ugspace.ug.edu.gh 128 6.2.3 Anti-Plasmodium parasite Antibodies in Immunized Mouse Sera by Microplate ELISA Microplate ELISA was conducted to screen sera of immunized BALB/c mice for anti- urinary and cultured Plasmodium parasite antibodies at the end of secondary immunization. Mice showing high antibody titre values in terms of optical density were selected and given a final booster of immunization with PAgHU before cell fusion. The steps for microplate ELISA were followed as described earlier (section 3.2.6), except that pre-immunization (normal mouse) sera were used as negative controls. 6.2.4 Feeder Cell Preparation and In Vitro Propagation of Myeloma Cells Feeder cells were used to condition the culture medium in order to provide an environment that ensured optimum growth and multiplication of myeloma cells retrieved from storage in liquid nitrogen at -196oC. Thymus gland was dissected out using a pair of dissecting scissors from a two-week old BALB/c mouse into a Petri dish. The dish contained 5 ml of Iscove’s Modified Dulbecco’s (IMDM) culture medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 50 µg/ml phosphate gentamycin prior to dissection. Dissection was done under aseptic conditions in a Clean Bench (Hitachi, Type PCV). The thymus gland was washed 3 times to get rid of blood in IMDM and then transferred into 0.5 ml of the medium. It was minced using the head of a sterile syringe plunger in order to free individual thymocytes. The freed thymus cells were re-suspended in a 50 ml Falcon centrifuge tube containing 10 ml medium and washed two times by centrifugation at 252 x g for 5 min at 37oC. The cells were re-suspended again after washing in fresh medium and then distributed into the wells of a 96-well plate at 100 µl per well. Myeloma cell lines X63/NS1/1-Ag4-1 (supplied by Biological Resources Center, Tokyo, Japan) were added to the same wells (100 University of Ghana http://ugspace.ug.edu.gh 129 µl/well) after washing and then cultured together in a humidified incubator conditioned with 5% CO2 in air. The wells with growing myeloma cells at the log phase were selected and expanded in large flasks. The growth of the cells was monitored using an inverted microscope (Olympus, CK30-F200, Japan). 6.2.5 Cell Fusion and Selection of Hybridomas BALB/c mice with the highest serum antibody titres were given a final booster of immunization as described in section 6.2.2 towards cell fusion. On the fourth day following the final booster immunization of mice, cell fusion was performed according to the methods described by Ansar and Ghosh (2013) and Pandey (2010) with modifications. The splenocytes of the most hyperimmunized mice were fused with myeloma cell lines using polyethylene glycol (PEG) as fusion agent. The spleen cells from hyperimmunized mice were prepared as described above as described for feeder cells in section 6.2.4 above. The cells were then similarly washed and resuspended in 10 ml PBS. An aliquot of the cell suspension was diluted with Trypan Blue (0.1% in PBS). The cells were counted using improved Neubauer Counting Chamber and the density determined using the formula stated below. Myeloma cells also in log growth phase were also washed in serum-free medium and the cell density determined. Where: X = the number of cells per ml ¼ = the mean of the 4 quadrants of the Neubauer chamber Cn = the total number of cells in the 4 quadrants of the Neubauer chamber 104 = is a constant Fd = the dilution factor of stained suspension and is defined as the ratio of the total volume of dilution to the initial volume of cell suspension The myeloma cells were mixed with spleen cells at a ratio of 3:1 myeloma to spleen cells, in a 50 ml tube. The mixed cell suspension was then centrifuged at 252 g for 5 min at X = ¼ x Cn x 104 x Fd University of Ghana http://ugspace.ug.edu.gh 130 37oC and the supernatant completely aspirated. The 50 ml tube containing mixed cell pellet was placed in a beaker (200 ml) containing water warmed at 37oC. One milliter (1 ml) of fusion solution [50% polyethylene glycol (PEG) 8000 and 5% dimethyl sulfoxide (DMSO) in PBS] also warmed to 37oC, was added drop wise to the cells over a period of 1min, while gently mixing the cells by gently shaking the tube in the beaker. The content was gently stirred with the same pipette for another 1min. Soon after, 2 ml of PBS was added over a period of 2 min while mixing as described above to dilute the PEG. A final 7 ml PBS was added over a period of 3 min and the suspension centrifuged as described before. Care was taken to ensure that the total time the cells spent in PEG solution alone did not exceed 8 min. The fusion products were then resuspended in 10 ml IMDM culture medium and washed by centrifugation as before. The final product was re-suspended in 20 ml culture medium and then distributed over two 24-well tissue culture plates at 500 µl/well. The plates were kept in the 37oC incubator conditioned with 5% CO2 in air as described above. On the following day, half of the medium in each well was aspirated out and a similar quantity of HAT-medium (IMDM supplemented with 10x10-4 M Hypoxanthine, 4x10-7 M Aminopterin and 1.6x10-5 M Thymidine) added to each well. The procedure was repeated on days 2, 4 and 6. Between days 8 and 13, wells with single large colonies of hybrid cells, as determined by observation under an Inverted Microscope were marked. The medium was allowed to become acidic (yellowish) and then screened by microplate-ELISA for the presence of antibodies. Hybridomas from selected wells were cloned while some stabilized as soon as possible. When hybridomas were growing well, (Plates 8 and 9 below) the HAT- medium was replaced with normal growth medium. Other cell fusions were performed using some of the remaining mice showing high serum antibody titers. University of Ghana http://ugspace.ug.edu.gh 131 6.2.6 Screening, cloning and cryopreservation of hybridoma cells Following cell fusion, culture supernatants were screened by microplate-ELISA for antibody activity as described in section 3.2.6 above. The microtitre plates were coated with either cultured P.falciparum parasite antigen (CPfAg) or Plasmodium infected human urinary antigen (PAgHU) at a concentration of 5.0 µg/well. Wells showing detectable antibody activity as revealed by optical densities were selected for cloning by limiting dilution. Some hybridomas, not cloned, were transferred into 6-well culture plates and allowed to grow and then cryopreserved. Cells (1x106/ml) were cryopreserved (-196oC) with 1ml culture medium supplemented with 5% (v/v) DMSO and 34% (v/v) FBS in vial tubes. Hundred microliters (100 µl) of cell suspension estimated to have 1 cell (ie 1 cell/ 100 µl \suspension), was added to each well and cultured. Supernatants from wells containing single cell colonies were finally screened again by microplate-ELISA for antibody reactivity. 6.2.7 Determination of cut-off point for ELISA negative reactivity results The absorbance ODs were measured at 414nm. Cut off point (Cf) for negative reactivity was calculated using the formula: Cf = Mean of negative controls + 2 x standard deviation (SD) of negative controls. Cf for plates coated with PAgHU = OD 0.0985nm; and for plates coated with CPfAg = OD 0.1345nm Table 10: Mode of grading of monoclonal antibody reactivity patterns Grading of OD Values Reaction with UPAg (ODs) Reaction with CPfAg (ODs) Negative Results Below 0.099 Below 0.135 Weak positive/ Trace (+/-) 0.099 – 0.499 0.135 to 0.499 Moderately strong positive (+) 0.500 – 1.499 0.500 – 1.499 Strong positive (2+) 1.500 – 2.499 1.500 – 2.499 Hyper positive (3+) 2.500 – 3.499 2.500 – 3.499 University of Ghana http://ugspace.ug.edu.gh 132 6.2.8 Immunoglobulin Classes of Monoclonal Antibodies in Hybridoma Culture Secretions The monoclonal antibodies (MAbs) produced in hybridoma cell culture supernatant were examined for immunoglobulin classes using the Pierce rapid isotyping kit (PRIK) for mouse sera (Thermo Scientific, Rockford, IL USA). This was done according to the manufacturer’s specification with modification. Briefly, the culture supernatant from each of the selected wells was diluted (1:5) by addition of 250 µl of PRIK sample buffer to 50 µl of the hybridoma cell culture supernatant in a 1.5 ml centrifuge (eppendorf) tube. 150 µl of the culture supernatant-buffer complex mixture was dispensed into the sample well of the PRIK cassette and then allowed to incubate for 15-20 minutes at room temperature (25oC). The test outcome indicated by the appearance of reddish-purple lines were read and graded visually by noting the immunoglobulin class label at the end of each line together with the intensity of coloration of the reddish-purple lines. The immunoglobulin class labels were IgG1, IgG2a, IgG2b, IgG3, IgM, and a control line labelled C; while the visual gradings were +, 2+, 3+ and 4+. 6.3.0 RESULTS 6.3.1 Immune Response in BALB/c Mice to Urinary and Cultured Plasmodium Antigens During immunization of BALB/c mice towards generation of MAbs, 15.0µg of urinary (PAgHU) and cultured (CPfAg) Plasmodium parasite antigens were used throughout the primary and secondary immunizations. Upon testing sera from the immunized mice for response to immunization, the results showed that only 20% (6/30) of the mice had a weak University of Ghana http://ugspace.ug.edu.gh 133 response (absorbance OD values ranged between 0.36 and 0.45, when measured at 414nm. Negative results had an absorbance of 0.12 and below) to the PAgHU. The majority of them showed no response at all. Also, the results showed that virtually all the mice exhibited no observable immune response to the CPfAg. Except for 10% (3/30) of them which exhibited a weak response (with absorbance ranging from 0.27-0.33; negative absorbance values ranging from 0.15 and below). Following the poor overall output of the initial immunizations, a fresh batch of 15 mice were immunized with 30.0µg (instead of 15.0µg) of the Plasmodium antigens and the results were presented in sections 6.3.2 and 6.3.3 below, as follows. 6.3.2 Detection of Anti-Urinary Plasmodium Antibodies in Immunized Mouse Sera by Microplate ELISA BALB/c mice immunized with 30.0µg of PAgHU antigens extracted from the urine of infected individuals and cultured parasites were tested for response to immunization by detection of anti-urinary Plasmodium antibodies in microplate ELISA. According the results (Figure 15), almost all the mice showed positive immune response to PAgHU. Mouse numbered 2 exhibited the highest immune response of 2.75 while mouse numbered 14 which apparently did not show any appreciable immune response at all, exhibited the lowest immune response OD of about 0.2. Also, mice numbered 11, 12 and 13 showed weak immune response with ODs below 0.3 from the beginning of immunization until the end of secondary immunization 1. However, after the end of secondary immunization 1, immune response to PAgHU increased in the mice until it had gone beyond OD 1.0 to OD 1.75 in mouse numbered 13. Finally, mouse numbered 4 after reaching immune response OD of 0.49 died at the end of secondary immunization 1 and therefore could not complete the immunization. University of Ghana http://ugspace.ug.edu.gh 134 Immune Response to Urinary Plasmodium Antigens Mse 1 Mse 1 Mse 2 Mse 2 Mse 5 Mse 6 Mse 6 Mse 6 Mse 6 Mse 8 Mse 8 Mse 8 Mse 11 Mse 11 Mse 12 Mse 12 Mse 13 Mse 13 Mse 13 Mse 14 Mse 15 Mse 15 0.000 0.500 1.000 1.500 2.000 2.500 3.000 I m m u n e R e s p o n s e ( O D s ) Stage of Immunization Mse 1 Mse 2 Mse 3 Mse 4 Mse 5 Mse 6 Mse 7 Mse 8 Mse 9 Mse 10 Mse 11 Mse 12 Mse 13 Mse 14 Mse 15 Figure 15: Immune Response of BALB/c Mice to Urinary Malaria Antigens University of Ghana http://ugspace.ug.edu.gh 135 6.3.3 Detection of Cultured Plasmodium Parasite Antibodies in Immunized Mouse Sera by Indirect ELISA As summarized in Figure 16 below, the results showed that unlike the response to PAgHU, almost all the mice exhibited weak or no immune response to CPfAg. Mice numbered 6, 11 and 13 however, showed good immune response to the CPfAg, with antibody response ODs being 0.79, 1.6 and 0.55 respectively. Consequently, these 3 mice were selected as the most suitable for cell fusion to generate the hybridoma cells for monoclonal antibody production. Figure 16: Immune Response in BABLB/c mice to Cultured Plasmodium Antigens Mse 1 Mse 6 Mse 6 Mse 11 Mse 11 Mse 12 Mse 12 Mse 13 14 Mse 14 0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 Im m u n e R es p o n se ( O D s) Stage of Immunization Mse 1 Mse 2 Mse 3 Mse 4 Mse 5 Mse 6 Mse 7 Mse 8 Mse 9 Mse 10 Mse 11 Mse 12 Mse 13 Mse 14 Mse 15 University of Ghana http://ugspace.ug.edu.gh 136 Plates 5 and 6 below are photographs of some of the microtitre plates after ELISA to determine immune response of BALB/c mice to urinary (PAgHU) and cultured (CPfAg) Plasmodium parasite antigens. The results corroborated what was shown by Figures 15 and 16 above that generally, immune response in the mice to the immunizing antigens increased with time. Also, the plates showed that before immunizations (columns 1 and 7), the mice were not showing response to any Plasmodium antigens, therefore, their sera reacted negatively to PAgHU and CPfAg in microplate ELISA. However, after primary immunizations with PAgHU and CPfAg, immune response to these antigens rose gradually until after secondary immunization 2 when immunizations had ended. Plate 5: A microtitre plate showing the response of mice 1 and 2 to urinary Plasmodium antigens as determined by microplate ELISA: Columns 1 & 7 = Response before immunization; Colum’s 2 & 8= Response after primary immunization; Columns 3 & 9 = Response after secondary immunization 1; Columns 4 & 10 = Response after secondary immunization 2; Wells A11 and A12 = positive control sera 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H University of Ghana http://ugspace.ug.edu.gh 137 Plate 6: A microtitre plate showing response of mice 3 & 4 to urinary Plasmodium antigens as determined by microplate ELISA: Columns 1 & 7 = Response before immunization; Colum’s 2 & 8= Response after primary immunization; Columns 3 & 9 = Response after secondary immunization 1; Columns 4 & 10 = Response after secondary immunization 2; Wells A11 and A12 = positive control sera 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H University of Ghana http://ugspace.ug.edu.gh 138 Plate 7 Plate 7: A cell fusion product on day-3 post cell fusion, showing a well on a 24-well tissue culture plate containing a mixture of unfused myeloma cells (MC; large cells that are degenerating), spleen cells (SC; small cells that are degenerating) and hybridoma cells (HC; large cells that are either dividing or are about to divide). Plate 8 Plate 9 Plate 8: Hybridoma cell colonies in various phases of growth on day 9 post cell fusion. Plate 9: Hybridoma cell colonies in various phases of growth on day 14 post cell fusion. MC (Myeloma cells) HC (Hybridoma cells) SC (Spleen cells) University of Ghana http://ugspace.ug.edu.gh 139 6.3.4 Characterization of Anti-Plasmodium Monoclonal Antibodies by Microplate ELISA In all 96 hybridoma cell clones were obtained after the 5th cell fusion to generate monoclonal antibody (MAb) secreting hybridoma cells. Analysis of these MAbs by microplate ELISA (Figure 17) showed that all the 96 clones secreted MAbs that were reactive to human urinary Plasmodium parasite antigen extract (PAgHU); and 66.8% (66/96) of these 96 hybridoma cell clones were also secreting MAbs that were reactive to cultured P. falciparum parasite antigens (CPfAg). Also, 31.3% of the MAbs produced were observed to be reactive to PAgHU only, 66.8% were reactive to both PAgHU and CPfAg and none was reactive to CPfAg alone. Figure 17: Reactivity of Anti- Plasmodium Monoclonal Antibodies by Microplate ELISA Moabs Reactive to PAgHU Only = 30 (31.3%) Moabs Reactive Both PAgHU & CPfAg 66 (66.8%) Moabs Reactive to PAgHU = 96 (100%) Total No. of Moabs Tested (n=96) Moabs Reactive to CPfAg Only = 0 (0.0%) University of Ghana http://ugspace.ug.edu.gh 140 6.3.5 Immunoglobulin Class and Reactivity of Monoclonal Antibodies by Rapid Isotyping Kit (PRIK) Table 11 below showed the results of the experiment to determine the immunoglobulin classes of selected hybridoma secretions (monoclonal antibodies), using the PRIK for mouse sera (Thermo Scientific, Rockford, IL USA). The results showed that the majority (8/14; 57.14%) of the hybridoma secretions characterized by isotyping contained monoclonal antibodies (MAbs) of the immunoglobulin IgM class (isotype) with very strong reactivity (4+ and 3+). The results also showed that 6/14 (42.86%) of the hybridoma cells were secreting the IgG class of MAbs. Of these number of hybridoma cells secreting the IgG class of MAbs, 3 cells were secreting the IgG1 subclass of MAbs with reactivity grades 4+, 3+ and 2+ respectively. Two cells were secreting IgG2a subclass of MAbs with strong (2+) reactivity; while one (1) was secreting the IgG3 subclass of MAbs with a moderately strong (+) reactivity. Finally, all the isotyping kit cassettes functioned normally, with the control line labelled “C”, appearing on each of the cassettes. Table 11: Immunoglobulin classes of selected hybridoma culture secretions Sample Serial Number (No) Monoclonal Antibody Sample Code Reactivity to Antigens Immunoglobulin Class Detected Isotyping Reactivity Strength PAgHU CPfAg 8 UCP1W8 1.57 (2+) 0.78 (+) IgM 3+ 12 UCP1W12 1.37 (+) 0.46 (+/-) IgM 4+ 13 UCP1W13 0.21 (+/-) 0.08 (-) IgM 4+ 30 UCP2W6 0.39 (+/-) 0.08 (-) IgM 4+ 31 UCP2W7 0.87 (+) 0.09 (-) IgG2a 2+ 32 UCP2W8 0.91 (+) 0.12 (-) IgG3 + 33 UCP2W9 0.36 (+/-) 0.08 (-) IgG2a 2+ 56 UCP3W8 2.82 (3+) 2.13 (2+) IgM 3+ 60 UCP3W12 2.98 (3+) 3.08 (3+) IgM 4+ 61 UCP3W13 0.60 (+) 0.60 (+) IgM 4+ 78 UCP4W6 0.62 (+) 0.18 (+/-) IgM 3+ 79 UCP4W7 1.73 (2+) 0.92 (+) IgG1 3+ 80 UCP4W8 2.87 (3+) 0.29 (+/-) IgG1 2+ 81 UCP4W9 0.92 (+) 0.28 (+/-) IgG1 4+ University of Ghana http://ugspace.ug.edu.gh 141 6.3.6 Cloning of Hybridoma Cells the Limiting Dilution Method Hybridoma cell colonies secreting monoclonal antibodies (MAbs) with highest antibody titres were cloned by the method of limiting dilution to obtain single clones of cells which were further expanded in order to obtain single clones of MAb producing cells. However, upon testing the culture supernatants for secretion of MAbs, the results were negative suggesting the absence of immunoglobulins. Attempts at further cloning of additional hybridoma cells, were not successful due to failure of the incubators to discharge carbon dioxide at the required concentration of 5% in air. The hybridoma cells were therefore cryopreserved to prevent them from dying, until the incubators are repaired. University of Ghana http://ugspace.ug.edu.gh 142 6.4.0 Discussion Since Kohler and Milstein invented the B-cell hybridoma technology for generating monoclonal antibodies (MAbs) in 1975, MAbs have become valuable molecular tools in both basic biochemical research and in medicine for disease diagnosis and treatment (Casadevall et al., 2004; Dozier, 2014). In order to obtain the antibodies required for diagnosis of malaria therefore, the experiments reported in this chapter were carried out with a main objective to develop MAbs against PAgHU. The MAbs were produced with the expectation that some would be useful in specifically detecting Plasmodium antigens in urine and therefore, serve as a promising tool for diagnosis of malaria in urine. The strategy was to immunize BALB/c mice with urinary (PAgHU) and cultured (CPfAg) Plasmodium antigens, and use immunized mice spleen cells in cell fusion to generate MAb against PAgHU and CPfAg. The in vitro culture hybridoma technology was adopted for production of MAbs in this study other than the in vivo ascites production technique. This was because, the ascites are often contaminated by other immunologically active compounds including viruses and bacteria that may be present in the mouse abdominal cavity (Dewar et al., 2005; Lipman et al., 2005), which compromise the specificity of the MAbs produced. Laboratory bred BALB/c mice were immunized for generation of MAbs instead of other animals such as rabbits, or hamsters, because the myeloma cell lines (NS-I and X63) available for cell fusion were from mouse origin. The decision to use myeloma cell fusion partners of the same species was based on the report by Grimaldi and French (1995) that “the myeloma cell fusion partner should be genetically compatible with the immunized spleen-cell source because hybridomas generated from cells of the same species are more stable than hybridomas generated from different species”. University of Ghana http://ugspace.ug.edu.gh 143 To obtain immunized murine spleen cells for cell fusion and generation of monoclonal antibodies (MAbs), laboratory bred BALB/c mice were immunized via the intraperitoneal route (IP) because it has several advantages over other routes which include the following. Firstly, the IP route is relatively more simple and easier and therefore, the most frequently and widely used channel for immunization of rodents (Shimizu, 2004; FSUIRAP, 2013). Secondly, it allows a wide distribution of immunogen to lymphoid tissues which leads to the induction of the heightened immune response (Bhowmick et al., 2009; Jerusalem and Eling, 1969), requisite for generation of highly reactive MAbs. In addition, even though it is comparable to the intravenous (IV) route of inoculation, in terms of the intensity of immune response induced, it is several times safer than the IV route. The IV route demands a great deal of caution and can lead to the death of most or all of the animals before the completion of immunization (Jerusalem and Eling, 1969). In spite of the numerous advantages of the IP route over other immunization roots, it has some disadvantages which include the risk of anaphylactic shock if booster immunogens are absorbed too rapidly in the vascular system. For these reasons therefore, the IP route was adopted for immunization of mice towards the generation of MAbs for development of the dipstick test for malaria. During the immunization of mice towards MAb development, the higher level of immune response induced by 30.0µg compared to 15.0µg of immunogen per mouse per immunization might be because 15.0µg of the crude antigen was not immunogenic enough. Indeed, it has been documented that the intensity of immune response that is mounted is partly dependent on the molecular size of antigen inoculated, such that a minimum of 5 kDa (8.30265 x 10-15 µg) of antigen is required to induce a minimum level of immunity; and the larger the size of the antigen University of Ghana http://ugspace.ug.edu.gh 144 the higher the immunogenicity (Varun, 2012). Therefore, the fact that 30.0µg of the crude antigens induced a more heightened level of immune response implied that it contained enough amount of antigen capable of stimulating humoral immune response in the mice against the malaria antigens. Also, the observation that mouse number 2 in the experiment showed the strongest immune response while those numbered 12 showed the weakest intensity of immune response even though they were all inoculated with relatively equal amounts of the immunogen (Figure 15) could be due to inherent genetic differences among them, which make some of them to exhibit better immunological response than others. In addition, the induction of a higher level of immune response in the mice by the urinary malaria antigens (PAgHU) compared to the cultured Plasmodium parasite antigens (CPfAg) (Figures 15 and 16) suggested that PAgHU was more immunogenic than CPfAg. In fact, a similar observation was made by Amanor et al. (1996), who reported that mice immunized with urine- based Schistosoma hematobium antigens exhibited a higher immune response than those immunized with S. haematobium soluble egg antigens. In all, 96 MAb-secreting hybridoma cell colonies were produced after cell fusion. The reactivity of all the 96 (100.0%) MAbs clones to PAgHU compared to only 68 (70.8%) which were reactive to CPfAgs supported the earlier statement that the former antigen was more immunogenic, and could therefore induce a higher immune response leading to the production of a greater number of reactive MAbs than CPfAg. Also, the detection of both PAgHU and CPfAg by 68 (70.8%) of the monoclonal antibodies suggests that these MAbs have a wider range of diagnostic applicability and could therefore be the most promising for development of the urine- University of Ghana http://ugspace.ug.edu.gh 145 based diagnostic test for malaria, provided that they are specific to malaria antigens only, and do not cross-react with any other antigens. In addition, the reactivity of 68 of the MAbs to both PAgHU and CPfAg suggests that the immunogenic factors that induced the production of these MAbs are shared in common by both antigens. Furthermore, that 30 (31.2%) of the MAbs could react with only PAgHU suggests that the immunogenic component of the malaria antigens that induced the production of such MAbs originated from PAgHU alone. Finally, the fact that none of the MAbs could react to only CPfAg implies that probably, none of the immunogenic factors of the antigens that induced the production of the MAbs came from CPfAg only. Antibodies are grouped into different immunoglobulin (Ig) classes known as isotypes which are characterized by differences in their biological features, including structure, distribution, function as well as affinity and specificity for different antigens. In view of this, the knowledge of the Ig classes of antibodies is useful in determining the probable nature of immune reaction that can occur between a particular MAb and the target antigen, and therefore helps in the selection of a given MAb for detecting particular disease antigen. Also, knowledge of the Ig class may be very useful in immunochemical engineering to improve upon the specificity and sensitivity of a selected MAb for diagnosis of a given disease. For these reasons, 7 out of the 96 MAb clones generated were examined for their Ig isotypes and strength of reactivity to the isotyping antisera, PAgHU and CPfAg. Only 7 MAbs were examined for Ig classes because, logistical challenges prevented the analysis of more than 7. The fact that almost all the 7 clones of MAbs examined had the IgM isotype compared to only 4 of them that had the IgG itotype suggested that the IgM producing spleen cells were more successful in fusing to myeloma cells than their counterpart IgG producing spleen cells. Also, the University of Ghana http://ugspace.ug.edu.gh 146 stronger reactivity (4+, 4+, 4+ 4+, 3+, 3+ and 3+) of the IgM isotype of MAbs compared to that (4+, 2+, 3+and 2+) of the IgG isotype, indicated that probably, there were more molecules of the IgM isotype than the IgG in each clone of MAb examined. Alternatively, it could be due to the fact that IgM being pentameric, had a higher specificity and affinity for the isotyping antisera than their IgG counterpart. Interestingly, however, in terms of the reactivity to PAgHU, the culture supernatant that contained more than one class/ subclass of the IgG isotype (i.e. IgG1 in addition to IgG2a, IgG2b and/ or IgG3) together with IgM, also had the highest reactivity for PAgHU, thereby suggesting that the presence of the IgG subclass istotypes, especially, IgG1 in the culture supernatant, heightens the reactivity of the MAb to PAgHU. Finally, in terms of the reactivity of the various classes of MAb to CPfAg, no clear reactivity pattern was observed between the Ig classes and CPfAg, thereby suggesting that the reactivity of MAbs to CPfAg is not influenced by Ig class characteristics. The single or monospecificity of monoclonal antibodies (MAbs) for their target antigens is necessary for differentiating between the target antigen and others, and forms a true basis upon which accurate diagnosis relies to control malaria by chemotherapy. The cloning of hybridoma cells to obtain a cell line originating from a single parental hybridoma cell is required to produce MAbs with monospecificity for target antigens. In order to generate MAbs with monospecificity for the PAgHU, therefore, the hybridoma cell colonies secreting multiple clones of MAbs with the highest antibody titre (ODs), were cloned by limiting dilution (Pandey, 2010) into single cells which were further multiplied in culture to obtain colonies of hybridoma cells capable of producing large amounts of MAbs. University of Ghana http://ugspace.ug.edu.gh 147 Upon testing the culture supernatants for reactivity to PAgHU and CPfAg, however, the results were negative, suggesting the absence of MAbs that were reactive to the target antigens. The observed absence of MAbs that were reactive to PAgHU and CPfAg in the culture supernatants, could however be due to abortion of antibody secretion by the monoclonal hybridoma cell lines. Indeed, these results confirms reports by several authors that monoclonal hybridoma cell lines sometimes abort MAb secretion, making them to function like myeloma cell lines. Attempts at further cloning by limiting dilution, however, were not successful due to failure of the incubators to discharge carbon dioxide at the 5% concentration in air needed for successful culture of the cells. The cultures were therefore suspended, and hybridoma cells were cryopreserved in liquid nitrogen to prevent them from dying until the incubators are repaired. University of Ghana http://ugspace.ug.edu.gh 148 CHAPTER SEVEN REACTIVITY OF MOUSE ANTI-PLASMODIUM MONOCLONAL AND POLYCLONAL ANTIBODIES IN ELISA AND WESTERN BLOTTING ASSAY 7.1.0 INTRODUCTION Polyclonal antibodies (PAbs) have several important uses as stated earlier in chapter two of this thesis report. In addition to these remarkable qualities, PAbs are the first kind of antibodies to be produced during infection and/ or immunization, and can give indication on whether a particular immunization exercise towards MAb generation has been successful or not. Therefore, in order to determine whether the immunization of mouse towards the generation of monoclonal antibodies as reported in this work, has been successful or not, PAbs produced against urinary and cultured Plasmodium parasite antigens were tested for reactivity against these antigens. Knowledge of the reactivity of the PAbs was necessary to select the best immune responder mice for cell fusion towards MAb Production. It was also necessary for characterization of the malaria diagnostic antigens present in infected human urine. In addition, the reactivity of the PAbs was required for optimization experiments to determine the best conditions for ELISAs and western blotting assays needed to characterize the MAbs being generated. In order to select the best hybridoma cells for cloning to generate MAbs specific to the urine-based malaria proteins, it was necessary to test the multiple clones of MAbs produced in hybridoma culture solutions for reactivity to the target urinary Plasmodium antigens. For these reasons, therefore, the experiments below were conducted to determine the reactivity of the monoclonal and polyclonal antibodies in ELISA and western blotting assays. University of Ghana http://ugspace.ug.edu.gh 149 Accurate diagnosis of malaria plays important roles in the managegent and control of the disease (Eibach et al., 2013). Also, it is necessary for the success of intervention programmes towards eradication of the disease (Eibach et al., 2013). The accuracy of diagnosis in turn, is largely dependent on the sensitivity and specificity of the tests used. This is because, a non- sensitive test would give rise to false negatives which could cause curative drugs not to be given to truly infected individuals who really need them. This factor could lead to complications and death in high risk groups (Reyburn et al., 2007). Also, a nonspecific test would result in false positives which could lead to dispensing of curative drugs to individuals who do not have the disease. Indeed, this factor is known to be the driving force behind parasite development of drug resistance (Reyburn et al., 2007). To ascertain whether the anti-mouse MAb and PAb produced were reacting accurately to the urinary and cultured Plasmodium parasite antigens, therefore, the sensitivity and specificity of these antibodies to the parasite antigens were determined using microplate ELISA and the result were compared to those of microscopy and other biomarkers of malaria. 7.1.1 Overall Aim To determine the reactivity of mouse anti-plasmodium monoclonal and polyclonal antibodies to different antigen preparations by microplate ELISA and western blotting assay Specific objectives  To determine the optimum conditions for conducting microplate ELISA experiments using MAbs and PAbs University of Ghana http://ugspace.ug.edu.gh 150  To determine the reactivity of mouse anti-Plasmodium monoclonal antibodies by indirect microplate ELISA  To determine the prevalence of Plasmodium infections in exposed individuals by mouse anti-Plasmodium UCP4W7 MAb microplate ELISA  To determine the prevalence of Plasmodium infections in exposed individuals by mouse anti-Plasmodium PAb microplate ELISA  To determine the reactivity of the UCP4W7 MAb to different antigen fractions by western blotting assay 7.2.0 MATERIALS AND METHODS 7.2.1 Optimum Conditions for ELISA Microplate ELISA experiments were carried out as described in section 3.2.5, to determine the best conditions, such as incubation temperature, time and type of microtitre plates for conducting microplate ELISA in this study. The findings were reported in the results section below. 7.2.2 Reactivity of Anti-Plasmodium Monoclonal Antibodies by Microplate ELISA The MAbs produced in hybridoma culture were tested for reactivity against the urinary Plasmodium antigen extract (PAgHU) and the cultured parasite antigen extract (CPfAg) using indirect microplate ELISA. Briefly the microtitre plates were coated with PAgHU (5 µg/ well) or CPfAg (5 µg/ well), and probed further with 100 µl of the supernatant from each well of the University of Ghana http://ugspace.ug.edu.gh 151 hybridoma cells in culture following cell fusion. The microplate ELISA experiment was then carried out as described in section 3.2.5 to determine the reactivity of the various MAbs to PAgHU and CPfAg. 7.2.3 Prevalence of Malaria in the Sample Population by MAb Microplate ELISA Microplate ELISA was carried out using one of the most reactive monoclonal antibodies (UCP4W7) as determined above, against direct unconcentrated urine samples from 420 individuals. This was done in order to compare the sensitivity and specificity of UCP4W7 with those of microscopy, quantitative buffy coat (QBC) test and polyclonal antibody ELISA. The 420 individuals were study participants whose blood samples had previously been examined by microscopy and QBC. The microtitre plates were coated (50 µl/ well) with urine sample preparation comprising 25 µl of urine from each participant and 25 µl of phosphate buffered saline (PBS, pH 7.2). The wells of the plate were coated overnight at 4oC, rinsed 2 times with excess washing buffer and then blocked (200 µl/ well) with blocking buffer comprising 5% w/v casein (Wako Pure Chemical Industries Ltd., Japan, Cat. No. 030-01505) in Dulbecco’s PBS (DPBS, pH 7.2) for 1hr. The wells were then incubated with the UCP4W7 MAb (100 µl/ well) for 2hrs at room temperature (25oC), flipped empty, banged on tissue paper to dislodge excess unbound MAb, and then washed 2 times with washing buffer. The wells were then incubated (50 µl/ well) with secondary antibody (goat anti-mouse polyclonal IgGAM conjugated with horse raddish peroxidase) (Abcam, Cambridge, UK; Cat No. ab6006), diluted (1:2000) in DPBS blocking buffer. The plates were flipped empty as before, banged and washed 5 times. ABTS [2,2 bis- azino-di-(3-ethylbenzthiazoline-6-sulphonate)] substrate solutions A and B were added (100 µl/ University of Ghana http://ugspace.ug.edu.gh 152 well), which gave a green colour reaction for positive results. Optical densities (ODs) of ELISA results were measured within 30 min using spectrophotometer (Multiskan Ascent Microplate Reader, Model 354; ThermoLab systems, Finland) at 414 nm. Cut off point (Cf) for negative reactivity was determined as described in section 6.2.7 above. After determination of the positive and negative results, the sensitivity and specificity of the UCP4W7 ELISA were determined using microscopy as a gold standard test. 7.2.4 Prevalence of Malaria in the Sample Population by anti-Plasmodium PAb Microplate ELISA Microplate ELISA to determine the reactivity of anti-Plasmodium PAbs to the 420 direct urine samples examined above was carried out as described for the UCP4W7 MAb above. Except for the PAb which was incubated (50 µl/ well) at 1:1250 dilution, all other aspects of the ELISA as well as the estimation of the relative sensitivity and specificity, were carried out as described in section 7.2.3 above. 7.2.5 Reactivity of Anti-Plasmodium PAbs to Different Antigen Preparations by Western Blotting Analysis Following the characterization of the proteins in the different antigen preparations by SDS-PAGE as described in sections 5.2.1 and 5.2.2 above, western blotting assay was used to determine the reactivity of the anti-mouse PAb to the different protein bands obtained as described in section 5.2.3 above. University of Ghana http://ugspace.ug.edu.gh 153 7.2.6 Reactivity of UCP4W7 MAb to different antigen preparations by SDS- PAGE and western blotting assay-1 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to characterize the proteins in different fractions of urine and cultured Plasmodium crude antigen extract as described in sections 5.2.1 and 5.2.2 above. Following the separation of proteins in different sample preparations by SDS-PAGE, western blotting assay was carried out to determine the reactivity of the UCP4W7 MAb to the various protein bands using the chemilluminescent detection method described by Mathews et al. (2009) with modification. After SDS-PAGE, the gel was washed in transfer buffer for 10 min, with gentle shaking. PVDF membrane about the same size as the gel (75 cm2) was pre-soaked in 100% methanol for 15 sec with gentle rocking and then transferred into the Invitrogen NuPAGE® Transfer Buffer (Life Technologies Corporation, California, USA) to soak for 5min with gentle rocking. The gel, the membrane and the pieces of blotting paper were assembled as illustrated in Figure 18 below, and then run for 1hr at 90mA. Negative electrode Blotting paper Gel PVDF membrane Blotting paper Positive electrode Figure 18: Mode of arrangement of the western blotting apparatus and gel system for western blotting analysis University of Ghana http://ugspace.ug.edu.gh 154 The membrane was washed with excess PBS for 5min after protein transfer and then washed again with PBST (0.05% Tween 20 in PBS) for 15min. The membrane was transferred into blocking buffer, made up of 3% bovine serum albumin (BSA) in PBST to block overnight at 4oC. Following blocking, the membrane was incubated with the primary antibody solution at 1:500 dilution of UCP4W7 MAb in blocking buffer for 1hr at 37oC with gentle rocking. The membrane was washed in excess PBS for 5min, and then in PBST for 15min with gentle rocking. The membrane was incubated with 1:2000 dilutions of the secondary antibody solution (horse raddish peroxidase conjugated goat anti-mouse polyclonal IgGAM antibody) in blocking buffer for 1hr at 37oC. The membrane after incubation was washed once more with excess PBS for 5min and in PBST for 15min. The membrane was then soaked in the immobilon western chemilluminescence reagent A and B (Millipore) with rocking for 5min to develop and then photographed using the Ez- Capture II ATTO Cooled CCD Camera System (ATTO Corporation, Japan) at 1min exposure. 7.2.7 Reactivity of UCP4W7 MAb to different antigen preparations by SDS- PAGE and western blotting assay-2 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to characterize the proteins in different fractions of urine, cultured Plasmodium crude antigen extract as well as antigens and vaccines of non-malarial parasites. The experiment was carried using the method described in sections 5.2.1 and 5.2.2 above. Following the separation of proteins in different sample preparations by SDS-PAGE, western blotting assay was carried out to determine the reactivity of the UCP4W7 MAb to the various protein bands obtained as described in section 7.2.7 above. University of Ghana http://ugspace.ug.edu.gh 155 7.2.8 Estimation of molecular weights of unknown proteins Following SDS-PAGE, the mobility (retardation factor, Rf) of the various protein bands in the gel, was calculated for the molecular weight (MW) protein standard as well as for the different antigen preparations, using the formula: Rf = d2 d1 Where Rf is the mobility (retardation factor) of the various proteins in the gel, di, measured in centimetres (cm), is the distance migrated by the tracking dye front, and is equivalent to the full length of the gel, d2 (cm) is also the distance travelled by the protein in the gel, measured from the top of the gel to the position of the band in the gel. A graph of the logarithm of molecular weight (log MW) against Rf was plotted and the line of best fit was drawn. Following this, the Rf of each protein band whose MW was to be determined was located on the x-axis and then followed vertically to the line of best fit from which the log MW of each protein was extrapolated. The antilog of the log MW value of each protein band was then taken to determine the approximate molecular weight (MW1) of the protein. Also, to improve the accuracy of the MW estimates, the equation of the line of best fit was determined as y = -1.1868x + 5.1538, where “y” represented the log MW and “x” was the Rf value for the protein. Using this equation, the Rf of each protein was substituted to determine the corresponding log MW of each protein and the antilog was read to obtain the second approximate University of Ghana http://ugspace.ug.edu.gh 156 molecular weight (MW2) for each protein. The final approximate molecular weight (MW) for each protein was then determined by taking the mean (MW1 + MW2/2) value of the two molecular weight readings estimated. 7.3.0 RESULTS 7.3.1 Optimum Conditions for ELISA to Ascertain the Reactivity of Anti- Plasmodium PAbs and MAbs Coating plates overnight at 4oC and 30 min at room temperature (22-25oC) gave similar ELISA outcomes (OD values). Coating of plates overnight at 4oC, however, had certain advantages. Firstly, it saves time. Enables trouble shooting to be carried out promptly for solution to unexpected results. Therefore, coating of plates overnight at 4oC was selected for carrying out all ELISA experiments. Also, coating urinary Plasmodium antigens on microtitre plates using carbonate- bicarbonate coating buffer (CCB) and PBS gave similar ELISA outcomes (ODs), however, PBS was selected as the routine buffer for coating of plates to run ELISA because of additional advantages over the CCB. These additional advantages are 1) PBS is the most abundant buffer in our laboratory, and 2) PBS is more readily available (obtainable) in our laboratory than CCB. Finally, upon comparing the immulon 2 and Nunc microtitre plates for conduct of the ELISA experiments, the immulon 2 plates gave a better output in binding Plasmodium antigens in terms of distinguishing between negative and positive control results. 7.3.2 Reactivity of mouse anti-Plasmodium MAbs to urinary and cultured parasite antigens by microplate ELISA-1 University of Ghana http://ugspace.ug.edu.gh 157 Table 12 showed the reactivity of different clones of mouse anti-Plasmodium MAbs to parasite antigens, PAgHU and CPfAg, extracted from the urine of infected individuals and cultured parasites respectively. Generally, the results showed that all the MAb clones were more reactive to the urinary Plasmodium antigen extract (PAgHU) than the cultured parasite antigen extract (CPfAg). According to the results, all the 48 (100%) MAb clones showed positive reactivity to PAgHU, but only 20 (41.7%) of them reacted positively to CPfAg. Of the 48 clones that reacted positively to PAgHU, 8 (16.7%) clones numbered 4 to 8, 26, 27 and 37; with code names UCP1W4 to UCP1W8, UCP2W2, UCP2W3 and UCP2W13 showed strong positive (2+) reactivity. 23 (47.9%) were moderate positivity (+), while the remaining 17 (35.4%) showed weak positive (+/-) reactivity. On the other hand, of the 20 clones that reacted positively to CPfAg, only 1 (2.1%) numbered 8, and coded UCP1W8 showed moderate positive reactivity. The remaining 19 (39.6%), clones showed weak positive reactivity to CPfAg. Finally, whereas all the MAb clones were reactive to PAgHU, 28 (58.3%) showed no reactivity to CPfAg. University of Ghana http://ugspace.ug.edu.gh 158 Table 12: Reactivity of anti-Plasmodium MAbs to urinary and cultured parasite antigen extracts-1 Sample Monoclonal Antibody Reactivity Sample Monoclonal Antibody Reactivity Absorbance OD (Grading) Absorbance OD (Grading) No Code PAgHU CPfAg No Code PAgHU CPfAg 1 UCP1W1 0.54 (+) 0.24 (+/-) 25 UCP2W1 1.43 (+) 0.09 (-) 2 UCP1W2 1.18 (+) 0.21 (+/-) 26 UCP2W2 1.95 (2+) 0.11 (-) 3 UCP1W3 1.01 (+) 0.38 (+/-) 27 UCP2W3 1.84 (2+) 0.10 (-) 4 UCP1W4 1.59 (2+) 0.18 (+/-) 28 UCP2W4 1.13 (+) 0.16 (+/-) 5 UCP1W5 1.51 (2+) 0.24 (+/-) 29 UCP2W5 0.47 (+/-) 0.08 (-) 6 UCP1W6 2.40 (2+) 0.43 (+/-) 30 UCP2W6 0.39 (+/-) 0.08 (-) 7 UCP1W7 1.92 (2+) 0.21 (+/-) 31 UCP2W7 0.87 (+) 0.09 (-) 8 UCP1W8 1.57 (2+) 0.78 (+) 32 UCP2W8 0.91 (+) 0.12 (-) 9 UCP1W9 1.20 (+) 0.15 (+/-) 33 UCP2W9 0.36 (+/-) 0.08 (-) 10 UCP1W10 1.39 (+) 0.18 (+/-) 34 UCP2W10 1.06 (+) 0.18 (+/-) 11 UCP1W11 1.23 (+) 0.11 (-) 35 UCP2W11 0.28 (+/-) 0.08 (-) 12 UCP1W12 1.37 (+) 0.46 (+/-) 36 UCP2W12 0.68 (+) 0.09 (-) 13 UCP1W13 0.21 (+/-) 0.08 (-) 37 UCP2W13 2.23 (2+) 0.14 (+/-) 14 UCP1W14 0.47 (+/-) 0.08 (-) 38 UCP2W14 0.29 (+/-) 0.08 (-) 15 UCP1W15 0.23 (+/-) 0.07 (-) 39 UCP2W15 0.53 (+) 0.09 (-) 16 UCP1W16 0.38 (+/-) 0.08 (+/-) 40 UCP2W16 0.54 (+) 0.08 (-) 17 UCP1W17 0.34 (+/-) 0.08 (-) 41 UCP2W17 0.31 (+/-) 0.24 (+/-) 18 UCP1W18 0.34 (+/-) 0.08 (-) 42 UCP2W18 0.29 (+/-) 0.08 (-) 19 UCP1W19 0.58 (+) 0.08 (-) 43 UCP2W19 0.75 (+) 0.08 (-) 20 UCP1W20 0.38 (+/-) 0.09 (-) 44 UCP2W20 1.00 (+) 0.21 (+/-) 21 UCP1W21 0.70 (+) 0.08 (-) 45 UCP2W21 0.77 (+) 0.08 (-) 22 UCP1W22 0.74 (+) 0.14 (+/-) 46 UCP2W22 0.55 (+) 0.13 (-) 23 UCP1W23 0.37 (+/-) 0.08 (-) 47 UCP2W23 0.57 (+) 0.13 (+/-) 24 UCP1W24 0.46 (+/-) 0.09 (-) 48 UCP2W24 0.30 (+/-) 0.19 (+/-) University of Ghana http://ugspace.ug.edu.gh 159 7.3.3 Reactivity of mouse anti-Plasmodium MAbs to urinary and cultured parasite antigens by microplate ELISA-2 Table 13 showed the continuation of the reactivity of different clones of mouse anti- Plasmodium monoclonal antibodies to parasite antigens, PAgHU and CPfAg, extracted from the urine of infected individuals and cultured parasites, respectively. Generally, these results also showed that all the MAb clones were more reactive to the PAgHU than to CPfAg. According to these results, all the 48 (100%) clones were reactive to both PAgHU and CPfAg. Of the clones that were reactive to PAgHU, 10 (20.8%) showed hyperpositive (3+) reactivity, 12 (25.0%) were strong positive (2+), 25 (52.1%) were moderate positive (+) and only 1 (2.1%) showed a weak positive (+/-) reactivity. Of the clones that were reactive to CPfAg, however, only 1 (2.1%) showed a hyperpositive reactivity. Two (4.2%) showed a strong positive reactivity, 16 (33.3%) were moderate positive, while the remaining majority of 29 (60.4%) clones were weak positive. University of Ghana http://ugspace.ug.edu.gh 160 Table 13: Reactivity of mouse anti-Plasmodium MAbs to urinary and cultured Plasmodium antigen extracts Sample Monoclonal Antibody Reactivity Sample Monoclonal Antibody Reactivity Absorbance OD (Grading) Absorbance OD (Grading) No Code PAgHU CPfAg No Code PAgHU CPfAg 49 UCP3W1 0.42 (+/-) 0.31 (+/-) 73 UCP4W1 2.74 (3+) 0.25 (+/-) 50 UCP3W2 1.77 (2+) 0.55 (+) 74 UCP4W2 2.86 (3+) 0.41 (+/-) 51 UCP3W3 1.67 (2+) 1.85 (2+) 75 UCP4W3 2.88 (3+) 0.21 (+/-) 52 UCP3W4 2.38 (2+) 1.29 (+) 76 UCP4W4 1.91 (2+) 0.54 (+) 53 UCP3W5 2.19 (2+) 0.51 (+) 77 UCP4W5 0.91 (+) 0.38 (+/-) 54 UCP3W6 2.80 (3+) 1.32 (+) 78 UCP4W6 0.62 (+) 0.18 (+/-) 55 UCP3W7 2.57 (3+) 0.53 (+) 79 UCP4W7 1.73 (2+) 0.92 (+) 56 UCP3W8 2.82 (3+) 2.13 (2+) 80 UCP4W8 2.87 (3+) 0.29 (+/-) 57 UCP3W9 1.98 (2+) 0.86 (+) 81 UCP4W9 0.92 (+) 0.28 (+/-) 58 UCP3W10 2.14 (2+) 0.69 (+) 82 UCP4W10 2.02 (2+) 0.39 (+/-) 59 UCP3W11 2.51 (3+) 0.46 (+/-) 83 UCP4W11 0.76 (+) 0.28 (+/-) 60 UCP3W12 2.98 (3+) 3.08 (3+) 84 UCP4W12 1.34 (+) 0.20 (+/-) 61 UCP3W13 0.60 (+) 0.60 (+) 85 UCP4W13 3.10 (3+) 0.56 (+) 62 UCP3W14 1.40 (+) 0.72 (+) 86 UCP4W14 0.67 (+) 0.26 (+/-) 63 UCP3W15 0.64 (+) 0.77 (+) 87 UCP4W15 1.43 (+) 0.25 (+/-) 64 UCP3W16 0.88 (+) 0.19 (+/-) 88 UCP4W16 1.11 (+) 0.18 (+/-) 65 UCP3W17 0.90 (+) 0.17 (+/-) 89 UCP4W17 0.50 (+) 0.74 (+) 66 UCP3W18 0.99 (+) 0.17 (+/-) 90 UCP4W18 0.78 (+) 0.27 (+/-) 67 UCP3W19 1.43 (+) 0.16 (+/-) 91 UCP4W19 1.30 (+) 0.16 (+/-) 68 UCP3W20 1.04 (+) 0.35 (+/-) 92 UCP4W20 1.70 (2+) 0.26 (+/-) 69 UCP3W21 1.67 (2+) 0.29 (+/-) 93 UCP4W21 1.31 (+) 0.52 (+) 70 UCP3W22 1.86 (2+) 0.45 (+/-) 94 UCP4W22 1.47 (+) 0.46 (+/-) 71 UCP3W23 0.84 (+) 0.31 (+/-) 95 UCP4W23 1.25 (+) 0.58 (+) 72 UCP3W24 1.16 (+) 0.25 (+/-) 96 UCP4W24 0.82 (+) 0.30 (+/-) University of Ghana http://ugspace.ug.edu.gh 161 7.3.4 Summary of the reactivity of mouse anti-Plasmodium MAbs to urinary and cultured parasite antigens Table 14 below summarized the reactivity of 96 mouse anti-Plasmodium MAb clones to PAgHU and CPfAg. As shown, the 96 MAb clones were more reactive to PAgHU than to CPfAg. Also, in terms of high reactivity strength, whereas 10/96 (10.4%) MAbs clones were hyperpositive (3+), 20 (20.8%) were strong positive (2+) and 48 (50.0%) were moderate positive (+) in reactivity to PAgHU. In contrast, only 1 (1.0%), 3 (3.1%) and 16 (16.7%) clones respectively, showed reactivity to CPfAg (Table 14). In relation to weak positive and negative reactivity, although 18/96 (18.8%) and no (0.0%) MAb clones were reactive to PAgHU more, 48 (50.0%) and 28 (29.2%) MAbs respectively, showed reactivity to CPfAg. Table 14: Summary of monoclonal antibody reactivity results Reactivity strength No of Monoclonal antibody clones reactive to Name Grade PAgHU No (%) CPfAg No (%) Hyperpositive 3+ 10 (10.4) 1 (1.0) Strong positive 2+ 20 (20.8) 3 (3.1) Moderate positive + 48 (50.0) 16 (16.7) Weak positive +/- 18 (18.8) 48 (50.0) Negative - 0 (0.0%) 28 (29.2) Total 96 (100.0) 96 (100.0) University of Ghana http://ugspace.ug.edu.gh 162 7.3.5 Reactivity of UCP4W7 MAb to different antigen preparations by SDS-PAGE and western blotting assay-1 Figure 19a showed the results of the separation of proteins in different antigen preparations by SDS-PAGE. As shown, PAgHU (lane 2) and the blood sample from a malaria negative individual from USA (lane 3) had more (>10) clearly visible protein bands each, than the rest of the various antigens separated by SDS-PAGE. These protein bands included those with estimated molecular weights (MW) of about 27.2, 32, 52 and 63.2 kDa. CPfAg (lane 4), had the next highest number (4) of clearly visible protein bands, which included proteins of MW 10.2, 13.5, 32 and 63.2 kDa. It was followed by the urine sample from a true malaria negative individual from Ghana (GH, lane 6), which also had 2 clearly visible bands of MW between 63.2 and 75.4 kDa. The malaria negative urine sample from JHU (lane 5), showed the least number of visible bands with an MW of about 13.5 kDa. The remaining antigens showed some bands which however, were not clear enough. Among this category of antigens was the Polio vaccine which showed bands that have joined together to form a smear. Lane 1 was occupied by the prestained SDS-PAGE broad range molecular weight marker Figure 19b, adjacent the coommassie blue gel electrophoregram, showed the results of the reactivity of the UCP4W7 MAb to the proteins separated by SDS-PAGE. As shown, the UCP4W7 MAb reacted with 10 protein bands in PAgHU, 4 of which were very sharp while the remaining 6 were faint (weak). The 4 sharp protein bands had MWs of about 27.2, 32, 63.2 and 75. 4 kDa. Also, UCP4W7 showed a high reactivity with 2 protein bands of MW 27.2 and 75.4 kDa in the cultured Plasmodium parasite antigen (CPfAg). The remaining antigens however, did not show any observable reactivity with the UCP4W7 MAb. University of Ghana http://ugspace.ug.edu.gh 163 Figure 19: a) Analysis of proteins in different antigen preparations by SDS-PAGE-1 and b) UCP4W7 probed western blotting assay-1. 1 = Molecular weight marker 2 = Human urinary Plasmodium antigen extract (PAgHU) 3 = Malaria negative blood sample 4 = Cultured Plasmodium falciparum crude antigen (CPfAg) 5 = Malaria negative urine from USA (JHU –Ve) 6 = Malaria negative urine from Ghana (GH –Ve) 7= Schistosoma mansoni soluble egg antigen (Sm. SEA) 8 = Schistosoma haematobium adult worm antigen (Sh. AWA) 9= Polio Vaccine 10= Measles vaccine 188 kDa 97 kDa 52 kDa 33 kDa 21 kDa 19 kDa 12 kDa 6 kDa F E D C a b University of Ghana http://ugspace.ug.edu.gh 164 7.3.6 Separation of proteins in different antigen preparations by SDS-PAGE and reactivity of UCP4W7 by western blotting assay-2 Figure 20a summarized the results of the separation of proteins in different antigen preparations by SDS-PAGE. As shown, with the exception of the JHU negative urine sample which had bands that were not distinguishable, all the proteins exhibited multiple bands after staining. Among the protein profiles with the multiple bands, the malaria negative urine sample from Ghana (GH-ve, lane 8), had the profile with the least number (2) of bands with estimated molecular weight (MW) being 63.2 and 75.4 kDa. CPfAg (lane 6), had 4 bands, 2 of which were clearly visible and the other 2 being faint. The 2 sharp bands were proteins of MWs being 13.5 and 63.2 kDa, while the weak bands had MWs of 10.2 and 32 kDa. The 2 direct urine samples (DUMIs) from the 3+ (DUMI-2, lane 2) and 4+ (DUMI- 1, lane 3) malaria positive individuals had the next highest number of bands. Each of these 2 samples with similar protein profiles had eight (8) visible bands, 4 of which were most prominent. These prominent protein bands had MWs being 27.2, 32, 45.6 and 63.2 kDa. The remaining samples comprising the PAgHU (lane 4), the malaria negative blood sample (MNBS, lane 5) and the yellow fever vaccine (lane 9), had protein profiles with the highest number of bands (>10) each. Of these, the yellow fever vaccine which apparently had the highest number of bands, had most of the bands within the upper part of the profile joining together to form a smear. Furthermore, the protein profile of the MNBS had a 13.5 kDa protein band (B) which was also present in the CPfAg and the yellow fever vaccine, but not in the profile of any other antigen preparation. In addition, MNBS had a 63.2 kDa band (E) which was also observed in all the other protein profiles. Finally, the results showed that the band sizes of proteins B and E were relatively bigger and darker than all the other bands observed in the entirety of the gel. Figure 20b showed the results of the reactivity of the UCP4W7 MAb with the bands in the protein profiles of the different antigen preparations as determined by western blotting assay. The University of Ghana http://ugspace.ug.edu.gh 165 results showed that generally, the UCP4W7 MAb was reactive to PAgHU, CPfAg, and DUMIs, but did not react with MNBS or the GH-ve and US-ve urine samples. The UCP4W7 MAb reacted specifically with more than 3 similar bands within the profiles of the DUMIs (lanes 2 and 3). The most prominent of these bands, labelled Z, with MW <6 kDa, was present only in the DUMIs, but not in PAgHU, CPfAg, MNBS, GH-ve or US-ve urine samples. The other prominent bands included a 27.2 kDa protein labelled C which was also present in CPfAg with strong reactivity, but in PAgHU with a weak reactivity (even though C showed a very strong reactivity in Figure 19b). This band was however not present in MNBS, GH-ve or US-ve. The last prominent band in the DUMIs that UCP4W7 reacted with was a 63.2 kDa protein labelled E, which was also present in PAgHU but not in either CPfAg, MNBS, GH-ve or US-ve. Also, the UCP4W7 MAb reacted with multiple bands within the profile of PAgHU. Three of these bands had the strongest reactivity and were protein D (32 kDa), a 63.2 kDa protein labelled E as well as a 75.4 kDa protein labelled F. Whereas protein D was detected in only PAgHU, F was also detected in only CPfAg. CPfAg also had 2 bands reacting strongly with the UCP4W7 MAb. These bands were the 27 and 63.9 kDa proteins, labelled C and F respectively and mentioned above. Furthermore, there were several bands within the yellow fever vaccine protein profile and one band in the molecular weight marker (lane 1), which reacted non-specifically with the UCP4W7 MAb. Lastly, there were no bands within the MNBS as well as GH-ve or US-ve that showed observable reactivity with the UCP4W7 MAb. University of Ghana http://ugspace.ug.edu.gh 166 Figure 20: a) Analysis of proteins in different antigen preparations by SDS-PAGE-2. and b): UCP4W7 probed western blotting assay-2. 1 = Molecular weight marker 2 = 3+ Malaria positive urine 3= 4+ Malaria positive urine 4 = Human urinary Plasmodium antigen extract (PAgHU) 5 = Malaria negative blood sample 6 = Cultured Plasmodium falciparum crude antigen (CPfAg) 7 = Malaria negative urine from USA (JHU –Ve) 8 = Malaria negative urine from Ghana (GH –ve) 9 = Yellow fever vaccine 3+ = 50-100,000 parasites/µL of blood 4+ = >100,000 parasites/ µL of blood 188 kDa 97 kDa 52 kDa 33 kDa 21 kDa 19 kDa 12 kDa 6 kDa E (63.2 kDa) A (10.2 kDA) B (13.5 kDa) Z (< 6 kDA) I (>111 kDa) H (>111 kDa) G (111.0 kDa) F (75.4 kDa) E (63.2 kDa) D (32.0 kDa) C (27.2 kDa) B (13.5 kDa) a b University of Ghana http://ugspace.ug.edu.gh 167 1 2 3 4 5 6 8 λ δ π 7.3.7 Detection of Plasmodium Proteins in Different Fractions of Urine and Plasmodium Antigens by Western Blotting Analysis Figure 20 showed the results of the reactivity of mouse anti-Plasmodium polyclonal antibodies (PAbs) with 6 of the antigen preparations resolved by SDS-PAGE in sections 5.2.1 and 5.2.2 above, using western blotting assay. As shown, the mouse anti-Plasmodium PAb reacted with bands in protein profiles of only 2 of the 6 antigen preparations analyzed. These antigen preparations were the PAgHU and the direct urine sample from a 3+ malaria positive individual (DUMI-2). The PAb reacted with the 50.9 kDa antigen (Ω) and the 54.6 kDa antigen (β) within PAgHU of which Ω was bigger and darker than β. Also, the PAb reacted with 3 bands within DUMI-1, which were the 27.2, 46 and 51 kDa proteins labelled π, δ and λ respectively. Furthermore, these results showed that the protein band λ was larger and darker than δ and π. Figure 21: Western blotting results Lane 1 Prestained broad range protein molecular weight marker Lane 2 Crude urinary Plasmodium antigen extract (PAgHU) Lane 3 Urine from Plasmodium negative individual from Ghana Lane 4 Urine from Plasmodium negative individual from USA Molecular Weight 175,000 80,000 58,000 β Ω 46,000 30,000 25,000 17,000 7,000 University of Ghana http://ugspace.ug.edu.gh 168 Lane 5 Cultured P. falciparum crude antigen extract (CPfAg) Lane 6 Plasmodium MSP119 antigen Lane 8 Urine from 3+ Plasmodium positive individual 7.3.8 Relationship between Molecular Weights of Standard Proteins and Their Mobility through the NuPAGE-MOPS Gel Table 15 below summarized the relationship of standard molecular weight proteins and their mobility (retardation factor, Rf) through the NuPAGE-MOPS gel as determined SDS-PAGE. As shown, the molecular weight (MW) of the standard proteins increased from a minimum of 6000 Da (6 kDa) to a maximum value of 188000 Da (188 kDa). The corresponding mobility (Rf) of the proteins on the other hand, reduced from a maximum of 0.94 to a minimum of 0.12. These results showed that the MW of the standard proteins were inversely related to their mobility through the gel. The results also showed that the MW of the various proteins were inversely related to the distance travelled through the gel such that the larger the MW of the protein, the shorter the distance travelled through the gel. Table 15: Relationship between the molecular weights of standard proteins and their mobility through NuPAGE MOPS gel Known Molecular weight (MW) of Standard Protein Bands (kDa) Molecular weight (MW) of Standard Protein Bands (Da) Log of MW of Standard Protein Bands Distance travelled in gel (D1) (cm) Retardation factor, Rf (D1/4.9) 1 6 6000 3.78 4.6 0.94 2 12 12000 4.08 4.4 0.90 3 19 19000 4.28 3.8 0.78 4 21 21000 4.32 3.5 0.71 5 33 33000 4.52 2.6 0.53 6 52 52000 4.72 1.8 0.37 7 97 97000 4.99 1 0.20 8 188 188000 5.27 0.6 0.12 University of Ghana http://ugspace.ug.edu.gh 169 7.3.9 Molecular Weights of Standard Proteins and Their Mobility through the NuPAGE-MOPS Gel Figure 21 below summarized the relationship between the molecular weight of standard proteins (Log MW) and the mobility of standard proteins in the gel. The results showed that the relationship between Log MW of standard proteins and the mobility Rf was linear, and the linear relationship could be determined by the equation (Y = -1.1868x + 5.1538). Figure 22: Molecular weights of standard proteins and their mobility through the NuPAGE-MOPS Gel y = -1.1868x + 5.1538 R² = 0.9983 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 0.00 0.20 0.40 0.60 0.80 1.00 L o g ( M o le cu la r w ei g h t/ d a lt o n s) Mobility (Rf) University of Ghana http://ugspace.ug.edu.gh 170 7.3.10 Estimation of Molecular Weights (MW1) of Unknown Proteins Tables 16, 17 and 18 below, showed the estimation of the molecular weights of the different proteins in the various protein profiles that were obtained by SDS-PAGE. As shown, the molecular size (MW) of each protein was inversely related to the distance travelled in the gel such that the larger the MW, the shorter the distance moved within the gel. Also, the MW of the proteins ranged from a minimum of 10.2 kDa for the protein labelled A, which moved the longest distance within the gel than all the other proteins. Protein G which moved the shortest distance among the proteins in the table had the largest molecular size. The tables also showed that apart from proteins A and B which had the same MW values as estimated by both extrapolation from the graph and calculation from the equation of the best line of fit, all the other proteins had different MW values when determined by the two different approaches. Table 16: Estimation of molecular weights (MW1) of unknown proteins Unknown protein bands Distance covered on gel (d2) Rf (d2/4.9) y-value (log MW) Antilog Estimated MW1 kDa A 4.7 0.96 4.01 10232.92992 10.2 B 4.2 0.86 4.13 13489.62882 13.5 C 3.0 0.61 4.44 27542.28703 27.5 D 2.7 0.55 4.51 32359.36569 32.4 E 1.4 0.29 4.79 61659.50019 61.7 F 1.1 0.22 4.86 72443.59601 73.2 G 0.4 0.08 5.03 107151.93052 107.1 University of Ghana http://ugspace.ug.edu.gh 171 Table 17: Estimation of molecular weights (MW2) of unknown proteins Unknown protein bands Rf (d2/4.9) y-value (log MW) antilog Estimated MW2 (kDa) A 0.96 4.01 10232.93 10.2 B 0.86 4.13 13489.6 13.5 C 0.61 4.43 26915.34 26.9 D 0.55 4.50 31622.78 31.6 E 0.29 4.81 64565.42 64.6 F 0.22 4.89 77624.71 77.6 G 0.08 5.06 114815.4 114.8 Table 18: Mean molecular weights of unknown proteins Unknown proteins Estimated MW1 (kDa) Estimated MW2 (kDa) Mean Estimated MW (kDa) A 10.2 10.2 10.2 B 13.4 13.7 13.5 C 27.3 26.7 27.2 D 32.1 31.6 32.0 E 62.1 65.3 63.2 F 73.2 77.2 75.4 G 107.4 114.0 111.0 7.3.11 Relative sensitivity and specificity of tests for different biomarkers of malaria Table 19 summarized the relative sensitivity (RSe) and specificity (RSp) of tests for different biomarkers of malaria using microscopy as the gold standard. As shown, generally, most of the tests had high specificities which ranged from 82.1% for fever to 99.9% for urobilinogen. Two of the tests, including those for proteinuria and CareStartTM malaria, however, had lower specificities which were 55.6 and 64.6% respectively. The test for anaemia had the lowest specificity which was below 50%.. Unlike the specificities, the sensitivities were rather very low, and mostly below 50%. On individual basis, the commercial University of Ghana http://ugspace.ug.edu.gh 172 rapid diagnostic test for malaria was very sensitive (RSe=96.6%) but not specific enough (RSp=64.6%) for diagnosis of malaria. Test for fever was fairly sensitive (RSe=61.0%) and specific (RSp=82.1%) in associating with malaria. The test for urinary proteins was also fairly sensitive (RSe=59.6%) and specific (RSp=55.6%) even though the specificity was lower than that of the test for fever. The tests for microhematuria (blood), leukocytes, bilirubin, urobilinogen, nitrite, ketones and glucose were not sensitive but very specific in detecting biomarkers of malaria compared to microscopy. Microscopy being the gold standard test, was assigned a relative sensitivity and specificity of (100%). The test for urobilinogen had the highest specificity (RSp=99.9%) while that for nitrite showed the lowest sensitivity (RSe=1.9%) in detecting biomarkers of malaria. Table 19: Sensitivity and specificity of tests for different biomarkers of malaria Test Number Tested Number Positive Number Negative Relative Sensitivity (%) Relative Specificity (%) Blood smear microscopy1 1262 587 675 100 100 CareStartTM malaria2 1262 697 (130) a 565 (129) b 96.6 64.6 Fever 1262 479 (121) a 783 (229) b 61.0 82.1 Anaemia 1262 655 (364) a 607 (296) b 49.6 46.1 Micro-hematuria 1262 121 (70) a 1141 (521) b 8.7 91.9 Proteinuria 1262 648 (298) a 614 (239) b 59.6 55.6 Leukocytes 1262 101 (66) a 1161 (523) b 6.0 94.5 Bilirubinuria 1262 27 (7) a 1202 (547) b 3.4 97.0 Urobilinogen 1262 88 (0) a 1174 (500) b 15.0 99.9 Nitrite 1262 21 (10) a 1203 (557) b 1.9 95.7 Ketones 1262 155 (57) a 1077 (472) b 16.7 89.6 Glucose 1262 19 (7) a 1227 (570) b 20 97.3 1 Gold Standard test 2 A commercial immunochromatographic (ICT)-based rapid diagnostic test for malaria a Number of samples that tested negative by Microscopy b Number of samples that tested positive by Microscopy University of Ghana http://ugspace.ug.edu.gh 173 7.3.12 Relative sensitivity and specificity of UCP4W7 ELISA and quantitative buffy coat test for malaria Table 20 illustrated the relative sensitivity (RS) and specificity (RSp) of UCP4W7 MAb and PAb ELISAs and QBC test for diagnosis of malaria using microscopy as the gold standard. The data showed that the UCP4W7 MAb-based ELISA was very sensitive (RS=96.9%) and highly specific (75.6%) in diagnosing malaria among study participants. The mouse anti-Plasmodium polyclonal antibody (PAb) ELISA, on the other hand, was more sensitive (RS=97.4%) than the gold standard microscopy, even though its specificity was poor (RSp=21.3%). The quantitative buffy coat test (QBC) was also sensitive (RS=88.7%) and fairly specific (RSp=66.2%), but relatively less efficient than UCP4W7 ELISA in detecting the disease among the participants. Blood smear microscopy being the gold standard test against which the other tests were evaluated, was assigned a relative sensitivity and specificity of 100%. Table 20: Sensitivity and specificity of UCP4W7 ELISA and QBC tests Test Number Tested Number Positive Number Negative Relative Sensitiv ity (%) Relative Specificity (%) Blood smear microscopy1 420 195 225 100 100 QBC2 420 245 (72) x 175 (26) y 88.7 66.2 UCP4W7 MAb ELISA3 420 240 (51) x 180 (10) y 96.9 75.6 PAb ELISA 420 366 (176) x 54 (6) y 97.4 21.3 1 Gold standard test 2 Quantitative buffy coat test for malaria 3 UCP4W7 monoclonal antibody enzyme linked immunosorbent assay x Number of samples that tested negative by Microscopy y Number of samples that tested positive by Microscopy University of Ghana http://ugspace.ug.edu.gh 174 7.4.0 Discussion The aim of the work described in this chapter was to determine the diagnostic applicability of the UCP4W7 MAbs and PAbs produced and described earlier in chapters 3 and 4 of this work, using microplate ELISA and western blotting assay. The rationale was to develop a promising MAb for specific detection of Plasmodium antigens in infected human urine to pave the way for the development of a urine-based diagnostic test for malaria. Determination of optimum conditions for conduct of microplate ELISA During the experiments to determine the optimum conditions for conducting microplate ELISA to ascertain the reactivity of PAbs and MAbs to urinary and cultured Plasmodium parasite antigens, coating of plates overnight at 4oC was selected and used for conducting all ELISA experiments rather than 30 mins at room temperature. Coating of plates for 30 mins required that antigens for coating were prepared in the morning, followed by dispensing into the plates before allowing them to incubate for 30 mins at room temperature so that the antigens would adsorb onto the wells. This mode of coating is time-consuming, and eventually, caused the time for ELISA, analysis of results, as well as trouble shooting for unexpected results, to extend late into the evening. During coating of plates overnight at 4oC, however, the preparation of antigens and incubation of plates were all done the previous day. Following the coating of plates, the ELISA experiment itself was carried out the following day without any interference. This option conferred several advantages over the first option, because, it allowed enough time to conduct the ELISA, and analyze the results. Also, in situations where the assay did not perform as expected, there was enough time to do trouble shooting. It was for these reasons therefore that coating of plates overnight at 4oC was used for all ELISA experiments rather than at room temperature for 30 mins. University of Ghana http://ugspace.ug.edu.gh 175 Coating of urinary Plasmodium antigens onto microtitre plates using carbonate- bicarbonate buffer (CBB, pH 9.6) and phosphate buffered saline (PBS, pH 7.2) gave similar ELISA outcomes (ODs). However, PBS was chosen and used for coating of plates for microplate ELISA because, PBS was the most abundant reagent obtainable in our laboratory, in that it was prepared in large quantities, was obtainable at all times and used at all times. Finally, upon comparing the immulon 2 and Nunc microtitre plates for conduct of ELISA, the immulon 2 plates gave a better output in binding Plasmodium antigens, in terms of their ability to distinguish between blank, negative and positive results. Consequently, the immulon 2 plates were selected and used for all ELISA experiments reported in this thesis. Reactivity of mouse anti-Plasmodium PAgHU and CPfAg by microplate ELISA The results showed that both the MAbs and PAbs were reactive to PAgHU and CPfAg (Tables 12, 13 and 14), as well as direct/ unconcentrated urine samples (DUMIs) from malaria infected individuals (Figures 18, 19 and 20), but with different reactivity strengths. The differential reactivity of the MAbs and PAbs to the antigen preparations mentioned could be due to the fact that the antigens that induced the expression and production of the antibodies had different immunogenicities. Also, it could be due to the fact that the Plasmodium antigens being reacted to in the different crude protein preparations, occurred in these protein extracts at different concentrations that affects the reactivity of the antibodies detecting them (Murray et al., 2008; Yzerman et al., 2002). Lastly, it could be because the different antibodies had different degrees of affinity to the antigens being reacted to in the different antigen extracts (Khor et al., 2013). Any of the factors stated above could have altered the biochemical activity of either the antigens, the antibodies or both, which therefore led to the patterns of differential reactivity observed in the ELISA experiments. University of Ghana http://ugspace.ug.edu.gh 176 The UCP4W7 MAb reacted in microplate ELISA with direct urine samples from microscopy positive and negative individuals (DUMIs). However, they did not react with urine from true malaria negative individuals from Ghana (GH-ve) and USA (US-ve). The inability of the UCP4W7 MAb to react with some of the microscopy positive urine samples could be due to the fact that they were false positives, as microscopy is capable of giving false positive results, as reported by (Ohrt et al., 2002; Rodulfo et al., 2007). On the other hand, it is possible that the antigens detected in the majority of the microscopy positive individuals were not expressed in strains of the parasites that infected the discordant microscopy negative individuals (Rosenthal, 2012; Gamboa et al., 2010). Also, the reactivity of UCP4W7 to some of the urine samples from microscopy negative individuals in ELISA (Table 16) could be due to the fact that those individuals were infected but failed to be detected by microscopy. This observation also confirms the findings by other workers that microscopy is not sensitive enough for accurate diagnosis of malaria (Steenkeste et al., 2010; Okell et al., 2009). In addition, the ability of the UCP4W7 MAb to react with PAgHU and CPfAg, but not with the true negative urine samples (GH-ve) and (US-ve) suggested that UCP4W7 would be capable of accurately distinguishing between malaria negative and positive individuals and therefore, promising for further exploration and development of a urine-based diagnostic test for malaria. Resolution of proteins in different antigen preparations by SDS-PAGE The observation of higher numbers of protein bands, by SDS-PAGE, in the profiles of PAgHU, DUMIs and MNBS, than the rest of the antigen preparations suggested that probably, PAgHU, DUMIs, and MNBS contained higher numbers of different proteins than the rest of the antigen preparations with fewer protein bands in their profiles. University of Ghana http://ugspace.ug.edu.gh 177 Also, the fact that the band sizes of proteins B and E in MNBS were larger and darker than those of the other proteins in SDS-PAGE suggested that probably, the relative abundance of B and E in MNBS was higher than those of the other proteins observed in the gel. Finally, the presence of several protein bands in PAgHU and the DUMIs but not in GH-ve and US-ve suggested that probably malaria infection led to the excretion of such proteins into the urine. This is particularly because, it is well established that malaria could induce changes in the urine of infected individuals (Sengupta et al., 2011; Basant et al., 2010). Reactivity of the mouse anti-Plasmodium polyclonal antibody (PAb) to different antigen preparations in western blotting assay The reactivity of the mouse anti-Plasmodium polyclonal antibody (PAb) to protein bands in PAgHU and DUMI-1, but not to any band within GH-ve and US-ve suggested that probably, those proteins were truly malaria antigens, hence their absence from the malaria negative urine samples. In addition, the lack of reactivity between the PAb and the Plasmodium MSP119 antigen suggested that that antigen was not among the Plasmodium proteins that induced the production of the PAb in the mouse. Furthermore, the reactivity of the PAb to one protein band each in both PAgHU and DUMI-2, with higher intensity and larger band size suggested that these proteins had higher relative abundance in the respective antigens preparations than the others as reported by Goins and Cutle (2000). Reactivity of the UCP4W7 monoclonal antibody to different antigen preparations in western blotting assay According to the results (Figure 19), the UCP4W7 MAb reacted in western blotting assay against PAgHU, CPfAg and DUMIs but not with MNBS, GH-ve and US-ve. These University of Ghana http://ugspace.ug.edu.gh 178 results confirm the suggestion made earlier that UCP4W7 was capable of accurately distinguishing between malaria positive and negative individuals. According to the results, a < 6 kDa protein (Z) and a 10.3 kDa protein (A) were detected by UCP4W7 in the DUMIs but not in PAgHU, CPfAg and the negative samples. This observation could be due to the fact that proteins Z and A did not originate from the Plasmodium parasite, but was a human metabolite that was synthesized in response to infection by the parasite, hence the inability to detect them in CPfAg. Conversely, it could be that probably, it was a Plasmodium parasite antigen but got lost in the course of extraction from the cultured parasites, hence its absence from CPfAg. Also, it has been reported elsewhere (Midgett and Madden, 2007) that the methods used in extraction of proteins from urine could lead to loss or modification of some antigens in the proteins. By this report, it could be inferred that probably, Z and A were urinary Plasmodium proteins, which were lost during extraction, leaving concentrations which were below the sensitivity that could be detected by UCP4W7. In fact, during extraction from urine, the antigens were dialyzed with a 10, 000 MW cut-off cellulose membrane as reported in the literature. Since protein Z for example, was smaller than the 10,000 MW dialysis membrane pore size, it was possible for some to be lost through dialysis, leaving a concentration that was too low to be detected in PAgHU but high enough in the DUMIs. This concentration, even though might be too low in PAgHU, probably might be highly immunogenic and therefore, was able to elicit the immune response that resulted in the production of antibodies against Z. These events could therefore, be the probable reasons for the ability of the UCP4W7 to detect this antigen in the DUMIs but not in PAgHU or CPfAg University of Ghana http://ugspace.ug.edu.gh 179 Furthermore, the detection of the 13.2 kDa antigen (B) and 32 kDa antigen (D) (Figures 19 and 20) by UCP4W7 in PAgHU, but not in either CPfAg or DUMIs or the negative samples suggested that probably, the strains of the Plasmodium parasites that produced antigens B and D in PAgHU, were different from the strains that constituted CPfAg and the DUMIs. This explanation was arrived at because urine samples from over 400 infected participants were pooled together in order to prepare PAgHU. Therefore, it was possible that these number of subjects were harbouring different strains of the parasites, some of which had certain antigens which are not shared in common by all the strains of the parasite. A critical observation of the western blotting results also revealed that UCP4W7 was reactive to a 27.2 kDa protein band labelled C, in CPfAg, PAgHU and the DUMIs, though the reactivity to PAgHU was weak. Protein C however, was not detected in GH-ve, US-ve and the true malaria negative blood sample. This reactivity pattern suggested that protein C was probably a Plasmodium protein that was excreted into the urine of infected humans, hence its presence in the cultured parasite antigen extract as well as the urine of infected individuals. Also, the fact that protein C was not detected in the Plasmodium negative human urine and blood samples suggested that probably C was not a human antigen. If these deductions were right, then UCP4W7 and antigen C together form a promising antibody-antigen pair for diagnosis of malaria in the urine of infected individuals. In addition, the reactivity of UCP4W7 with the 63.2 kDa protein (E) in both PAgHU and DUMIs, but not in CPfAg or the negative samples, suggested that probably, antigen E was an RBC membrane bound Plasmodium protein that was lost through the removal of RBC membranes during preparation from the cultured parasites. On the other hand, it could be that E was an anti-malaria human metabolite that was produced by the host only after infection. University of Ghana http://ugspace.ug.edu.gh 180 These could therefore be the possible reasons for the observed reactivity to E in PAgHU and DUMIs but not in CPfAg and the negative samples. The detection of the 75.4 kDa protein band labelled F with reducing reactivity strength from PAgHU to CPfAg to DUMI-2 (>100,000 parasites/ µl of blood) and then to DUMI-1 (50,000 - 100,000 parasites/ µl of blood), suggested that antigen F was a true Plasmodium parasite protein whose concentration in the urine was probably associated with parasite density. This suggestion was confirmed by the fact that the reactivity of UCP4W7 to F in PAgHU was stronger than that observed in the other antigens. PAgHU was concentrated from several Plasmodium positive urine samples and probably had a higher amount of the antigen than the other samples. The reduction in reactivity to F with parasite density therefore suggested that antigen F could probably be very promising for estimation of parasite density in infected individuals. Furthermore, the fact that UCP4W7 detected 3 protein bands (G, H and I) of molecular weight from 111.0 kDa and above as well as proteins B and D in PAgHU but not in CPfAg, the DUMIs, GH-ve, US-ve or MNBS, suggested that probably, those antigens were Plasmodium proteins, which however, were not common to all the parasites detected in the infected individuals. In respect of this, it was reported that some of the P. falciparum parasites in Africa and Peru for example, do not express histidine-rich-protein 2 (HRP2) because they lack the gene for expression of the antigen (Gamboa et al., 2010). Rosenthal (2012), also reported that a certain fraction of P. falciparum parasites in Africa express a modified form of the HRP2 antigen, whiles, other malaria parasites like P. malariae, P. vivax, P. ovale and now P. knowlesi, do not express the HRP2 antigen at all, and therefore, any test directed at this antigen would be negative, and would not necessarily mean that the person did not have malaria. On the other hand, owing to the fact that the DUMIs were direct unconcentrated urine University of Ghana http://ugspace.ug.edu.gh 181 samples, it was also possible that they contained the said antigens but at concentrations lower than the sensitivity threshold of UCP4W7, hence the inability to detect them in the DUMIs. The reactivity of UCP4W7 to the proteins E (63.2 kDa) and F (75.4 kDa) in PAgHU in western blotting assay with a very high intensity and larger band sizes than the other proteins also confirmed the suggestion that probably those proteins had higher relative abundance in PAgHU than all the other antigen preparations. Relative sensitivity and specificity of tests for different biomarkers of malaria against blood smear microscopy as the gold standard test To evaluate the sensitivities and specificities of the various tests for diagnosis of malaria, microscopy was used as a gold standard test against which the various tests were measured. This was because, microscopy is presently the standard diagnostic tool of measure for determining the accuracy of performance of other diagnostic tests (Eibach et al., 2013; Rosenthal, 2012; Reyburn et al., 2007). The determination of the relative sensitivities and specificities of the tests for various biomarkers of malaria compared to microscopy showed that tests for ketones, microhematuria, leukocytes, nitrites, glucose, bilirubinuria urobilinogen as well as PAb ELISA, were highly specific but had very low sensitivities. The reactivity patterns of these tests suggest that they would not be very good for accurate diagnosis of malaria, since they could result in high false negative rates which could cause curative drugs not to be given to truly infected individuals who really need them (Murray et al., 2008). Also, the tests for fever and proteinuria, even though their relative sensitivity and specificity were above 50% they would not be very accurate or useful in diagnosis of the disease because, for a test to be clinically useful, the combined relative sensitivity and specificity should not be less than 170% (Wians, 2009). University of Ghana http://ugspace.ug.edu.gh 182 The estimation of the relative sensitivity and specificity of the UCP4W7 MAb (Table 16) showed that its combined relative sensitivity and specificity (96.9% + 75.6%) was 172.5%. The fact that its combined reactivity was greater than 170% showed that this test was accurate enough for distinguishing between malaria infected and uninfected individuals (Wians, 2009) and therefore, promising for further development of a urine-based test for diagnosis of malaria. Also, even though this combined reactivity was more than 170% it was lower than the expected ideal of 198%. This shortfall however, could have been because microscopy is not accurate enough to be used as a gold standard test (Alemu et al., 2014). A correction of these microscopy results by the polymerase chain reaction (PCR) technique as performed by (Eibach et al., 2013; Hopkins et al., 2008), could have reduced the rates of false negatives and positives, and consequently raised the combined reactivity of the UCP4W7 ELISA far above the observed 172.5%. Confirmatory tests by PCR to correct the microscopy results, however, were not carried out because of lack of logistics. The quantitative buffy coat (QBC) test unlike the UCP4W7 ELISA, had fairly high relative sensitivity and specificity but a lower combined reactivity (154.9%) than the 170% threshold required for accurate diagnosis. Therefore, for it to be used as a reliable test for diagnosis of malaria, especially, in situations like drug sensitivity studies, its sensitivity and specificity need to be corrected by PCR as done presently for blood smear microscopy (Eibach et al., 2013; Hopkins et al., 2008). Finally, the higher sensitivity (97.4%) and extremely lower specificity of the PAb ELISA compared to microscopy suggested that probably, it would not be accurate for diagnosis of malaria in the urine of infected individuals, even when microscopy results are corrected. University of Ghana http://ugspace.ug.edu.gh 183 CHAPTER EIGHT GENERAL DISCUSSION AND CONCLUSIONS Since the identification of malaria as an important global health problem, several efforts have been made to control the disease. These include attempts to eliminate vector Anopheles mosquitoes by insecticides, destruction of breeding places of mosquitoes in order to break disease transmission cycle as well as the elimination of causative Plasmodium parasites by anti-malarial drugs. Nevertheless, these efforts have not achieved the expected goal of disease eradication (Jain et al., 2014). As a result, the prevalence of the disease is still high, especially, in sub-Saharan Africa, where disease burden and death toll are greatest (Jain et al., 2014). Malaria eradication by chemotherapy requires accurate identification of infected individuals in endemic populations, which therefore demands the utilization of fast, simple, sensitive and specific diagnostic tools in order to achieve desired results. Currently, blood smear microscopy is the gold standard test for routine and confirmatory diagnosis of malaria (Eibach et al., 2013, Azikiwe et al., 2012). Also, immunochromatographic tests (ICTs), popularly known as rapid diagnostic tests (RDTs), as well as tests based on the use of clinical signs and symptoms, are some of the alternative tests for routine diagnosis of the disease (Eibach et al., 2013, Azikiwe et al., 2012). However, all of these tests are not accurate enough, they are labour-intensive and/ or invasive which therefore limit their effectiveness for control of the disease (Alemu et al., 2014; Steenkeste et al., 2010; Okell et al., 2009). For these reasons, it has become necessary to develop new tests University of Ghana http://ugspace.ug.edu.gh 184 which are more sensitive and specific, faster and non-invasive for improved diagnosis of malaria. The identification of parasite antigens and anti-parasite metabolites or antibodies in tears, saliva and urine of infected humans (Nolen et al., 2013; Ayong et al., 2005), highlighted the importance and the possibility of using such metabolic fluids for development of non- invasive tools for malaria diagnosis. Urine, in particular, was considered in this study as the most reliable excretory/ secretory metabolic by-product for development of non-invasive diagnostic tools for malaria because of the following reasons. Firstly, it is the only body fluid which can be obtained readily in large quantities non-invasively (Decramer et al., 2008). Secondly, urinary proteins are more stable to harsh environmental conditions than other biofluids such as the widely used serum and plasma (Nolen et al., 2013; Decramer et al., 2008). Also, it is ethically easier to sample urine than blood, which is equally, a rich source of biomarkers (O’Riordan et al., 2007). Lastly, various researchers have reported the detection of Plasmodium antigens and anti-malarial human metabolites in the urine of infected individuals (Thomas et al., 2010; Howard et al., 2007; Katzin et al., 1988; Rodriguez-del Valle et al., 1991). For these reasons, this study directed efforts at producing a mouse anti-malarial MAbs that could be used to develop a rapid tool for diagnosis of the disease through a MAb-based detection of parasite antigens and/ or anti-malarial metabolites in the urine of infected individuals. In the studies described in chapter four, malaria infected and uninfected subjects were identified from the endemic population using blood smear microscopy, quantitative buffy coat (QBC) test and commercial rapid diagnostic test (RDT) kits. These studies were carried out partly with a view to obtain infected urinary antigens for immunizations towards MAb University of Ghana http://ugspace.ug.edu.gh 185 generation. Also, the studies were conducted in order to select true negative control urine samples for ascertaining the specificity of the MAb-based tests to be developed. The successful discrimination of infected from uninfected subjects among the study population enabled the successful extraction of malaria related antigens for generation of the anti-malarial MAbs. A high malaria prevalence of 46.5% was found among the study population, which was not significantly different from the 52.0% prevalence obtained in a pilot study conducted 9 years earlier in the same community. These findings confirmed that reported by Jain et al. (2014), who stated that in areas with stable transmission in sub-Saharan Africa, malaria prevalence could be as high as 52.0%. It also confirmed the statement by some writers that even though several efforts have been put in place to control malaria, their overall impact has been lower than expected (Eibach et al., 2013; Reyburn et al., 2007; Shiff, 2002). These results therefore justified the need for new intervention tools to intensify efforts towards the eradication of the disease. According to the studies, malaria was significantly associated with sex, age, and fever, but not with anaemia. The occurrence of higher prevalence of malaria among females in these studies, even though it confirmed what Ayele et al. (2012) have reported, was suspected to be linked to behaviour more than to sex, as reported by other authors such as Steketee et al. (2001). The studies reported in chapter four, however, lacked the immunological and biochemical analyses needed to ascertain the link between malaria and sex. Therefore, it was not concluded that the higher prevalence of malaria obtained in females than males was sex- linked. However, the fact that no conclusive agreement have been reached by researchers on this issue, showed clearly that further studies are required to clarify the relationship between malaria and sex. University of Ghana http://ugspace.ug.edu.gh 186 Authors such as Griffin et al. (2014) and Schellenberg et al. (2004) have reported that understanding the relationship between malaria burden and age is important in selecting high risk groups in which priority intervention measures could be focused in order to prevent malaria catastrophe. Therefore, the observation of significantly higher prevalence of malaria and higher parasite density among children than adults in these studies were important findings that confirmed reports by other writers that children are more susceptible to the disease because they lack the immunity against the disease. These studies have therefore consolidated the findings that children are among the topmost priority group on which targeted intervention measures should be focused in order to minimize the disease burden in endemic populations. The observation of the highest parasite density among children as revealed by these studies has also shown that children are the group to concentrate on in order to obtain malaria antigen-rich urine samples for further studies. The ability of these studies to determine the association between malaria and fever as reported by other writers has also corroborated the findings made. Anaemia is known to be triggered by a variety of factors which include parasitic infections and malnutrition (Santana-Morales et al., 2013). Therefore, the fact that anaemia could not be linked to malaria in the studies described in this work, confirmed that probably, other factors could be involved in the anaemia observed among the study population. The involvement of other factors in the anaemia observed, however, was not investigated because that was not part of the study objectives. In studies described in chapter five of this write-up, P. falciparum species-specific HRP2 and Plasmodium pan-specific antigens were detected in the urine of infected individuals by the commercial blood-based RDT kits. These findings provided useful information on the potential of urine as a reliable source of biomarkers of malaria. Also, they University of Ghana http://ugspace.ug.edu.gh 187 indicated that urine is a promising candidate for development of non-invasive rapid tests for diagnosis of malaria. In the SDS-PAGE experiment to characterize biomarkers of malaria from infected human urine (chapter five), the number of protein bands observed in the profiles of infected urine samples decreased with parasite density in infected individuals. This observation suggested that probably the urine of infected individuals would serve as a good material for determination of efficacy of antimalarial drugs. This finding has promising implications particularly, for antimalarial drug developers. It demonstrated that urine of infected subjects could constitute a better alternative for studies on anti-malarial drug efficacy than blood, because, it is ethically easier to sample urine than blood (O’Riordan et al., 2007). The detection of some proteins in the profiles of infected urine samples at high concentrations by SDS-PAGE and western blotting assay (chapter five), also supported reports that urine samples from infected subjects contained adequate amount of proteins for studies on such malaria-related biomarkers (Thomas et al., 2010). In those experiments, the mouse anti-Plasmodium PAb detected 2 proteins of MW 50.9 and 54.6 kDa in PAgHU and 3 proteins with MW being 27.2, 46.0 and 52.7 kDa in DUMI-2. This observation indicated that those proteins could be potential urine-based diagnostic markers of malaria. Also, the strong reactivity of the 50.9 kDa protein (Ω) in PAgHU and 52.7 kDa protein (λ) in DUMI-2 to the PAb, showed that probably, those proteins would induce the production of MAbs with very high sensitivity. The fact that the 2 proteins had thicker bands as detected by the PAb, showed that those proteins had higher relative abundance or concentrations in their respective antigen preparations. In their quantitative analysis of the complex protein mixtures, Gygi et al. (1999) reported that measured differences in protein expression in yeast correlated with its metabolic function under glucose-repressed conditions. Since the level of University of Ghana http://ugspace.ug.edu.gh 188 expressed proteins is directly related to the concentration or the relative abundance of that protein in a biological sample, it implied that the proteins Ω and λ which showed thicker bands compared to the others, probably, play more important biological roles within the Plasmodium parasite (McAfee et al., 2006). Indeed, it is also possible that the higher immunogenicity of the 2 proteins as observed in the studies was caused by their higher concentrations in the urine samples. On the other hand, the concentration of proteins in a urine sample at a given time is known to reflect the resistance of such proteins to the degradation caused by factors such as handling and storage conditions as well as the proteolytic enzyme activity that is ongoing in the sample (Pisitkun et al., 2006). In relation to this report, therefore, it is possible that the 2 proteins which exhibited thicker bands, as observed above, were more stable to the degradative effect of the factors mentioned above. The concentration of proteins in urine has also been reported to reflect the ability of such proteins to pass through the filtration membranes within the glomerulus (Nolen et al., 2013). In relation to this therefore, the higher concentrations of the 2 proteins in the urine samples as revealed by their thicker band sizes, indicated that possibly, such proteins had greater ease of crossing the glomerular barrier into the urinary bladder than the others. This finding, therefore, has important implications for further studies on such proteins in urine. Finally, the proteins Ω and λ appeared to have important biological functions in the Plasmodium parasites as revealed by their strong reactivity to the PAb, and thicker band sizes that were suggestive of high relative abundance in the urine samples. The veracity of these observations however were not ascertained by the studies reported in this write-up. It is therefore suggested that further studies should be conducted to investigate the importance of these proteins to the Plasmodium parasite and the implication for diagnosis and control of the infection due to these parasites. University of Ghana http://ugspace.ug.edu.gh 189 In the study to determine the association of malaria, haemoglobin levels and anaemia with urinary hepcidin levels in malaria infected subjects (chapter five), the highest prevalence of malaria and parasite density were observed among children, while the lowest prevalence of the disease and parasitaemia were observed among pregnant women. Those observations confirm the findings that children were among the group of individuals at the highest risk of infection with malaria (Genton et al., 2008). The rather low prevalence of the disease and parasitaemia in the pregnant women who are also among the highest risk group, however, might be attributable to the fact that they were undergoing intermittent preventive treatment (IPT), as part of the Ghana Ministry of Health/ Ghana Health Service and the National Malaria Control Programme policy on management of malaria among pregnant women. Also, the lowest haemoglobin level and anaemic status observed among the children, even though they had the highest disease burden among the cohort of participants examined confirmed the findings that anaemia among affected individuals might be attributable to several factors, including malnutrition and hookworm infection rather than to malaria alone (Santana-Morales et al., 2013). Increased levels of hepcidin was observed in the urine of malaria infected individuals which was significantly associated with parasitaemia but not haemoglobin levels or anaemia. This observation demonstrated that urinary hepcidin would be a promising biomarker for diagnosis of malaria. It also indicated that perhaps, changes in urinary hepcidin levels with parasitaemia could be explored for development of a quantitative diagnostic test for malaria. In addition, the decreasing levels of urinary hepcidin with parasitaemia, would be very useful in the evaluation of anti-malaria drug efficacy. The apparent lack of association between urinary hepcidin and haemoglobin levels or anaemia, however, suggested that urinary hepcidin levels would not be a useful biomarker for determination of haemoglobin levels or anaemia. Probably, this apparent lack of association University of Ghana http://ugspace.ug.edu.gh 190 might have been caused by the fact that several confounding factors combined together to give rise to the haemoglobin level or anaemic status of an average individual. Further studies are therefore, required to characterize the longitudinal variation in urinary hepcidin levels with changes in haemoglobin levels and anaemia in the presence or absence of malaria, with or without other concomitant infections which are also implicated in the blood haemoglobin levels or anaemic status of an individual. In chapter six of this thesis, MAbs were generated by cell fusion, using hybridoma technology. The ascites production method were not used because of the possibility of contamination by unwanted immunologically active compounds in the peritoneum of the mice, which might interfere with the specificity of the MAbs generated as reported (Dewar et al., 2005; Lipman et al., 2005). It has been reported that MAb-secreting hybridoma cells generated using spleen cells from the same species of animal as the myeloma cell line oringin, are more stable than those generated from different species of fusion partners (Grimaldi and French, 1995). In the immunization experiments towards MAb generation, therefore, BALB/c mice were used because, the myeloma cell lines that were available for cell fusion were of mouse origin. Also, the route of immunization of laboratory animals towards antibody production has been reported to be very instrumental in the quality of immune response and the corresponding sensitivity of the antibodies generated (Mohanan et al., 2010). In this study therefore, BALB/c were immunized via the peritoneal route because it was relatively easier, safer and could lead to the induction of the high immune response needed for generation of MAbs with high sensitivity. It was therefore not surprising that at the end of the immunization experiments, only one (6.67%) out of the 15 mice finally immunized, died, before obtainment of the last immune sera for determination of immune response in the mice. In addition, the University of Ghana http://ugspace.ug.edu.gh 191 high reactivity of the immune sera and the MAbs produced to PAgHU could probably have been contributed by the route of immunization used. Furthermore, in those experiments, the fact that one mouse did not show any immune response at all, suggested that it might have been immunologically suppressed. Indeed, this observation confirmed the reports that there are immunologically suppressed individuals in each population who may not respond positively to invasion by foreign antigens (Kopel et al., 2012). The potential usefulness of the MAbs generated as a promising diagnostic reagent for malaria was demonstrated through their reactivity with both urinary (PAgHU) and cultured Plasmodium (CPfAg) antigen extracts. In those experiments, all the 96 clones (100%) of the MAbs reacted with PAgHU, 70.8% reacted with both PAgHU and CPfAg, (31.3%) reacted with PAgHU only, while none reacted with CPfAg alone. This observation was very interesting because, the reactivity of some of these MAbs to either PAgHU only, or to both PAgHU and CPfAg, indicated that perhaps, the MAbs were either species-specific, pan- specific or both. The reactivity also indicated that probably, the MAbs would also have a wide range of applicability. These species-specific and pan-specific qualities might have been induced by PAgHU as demonstrated in chapter 5 of this report that PAgHU contained both the P. falciparum species-specific HRP2 and pan-specific LDH antigens. In view of this, the reactivity of all the 96 clones of the MAbs to PAgHU confirmed the report that the urine of infected individuals would be potentially useful as a promising source of biomarker for diagnosis of malaria. Finally, these results demonstrated that MAbs generated against malaria antigens in urine of infected individuals would be very useful in development of a tool for diagnosis of the disease globally. University of Ghana http://ugspace.ug.edu.gh 192 In chapter seven, the reactivity of mouse MAb and PAb produced against PAgHU and CPfAg as well as direct urine samples from study participants were determined. Both MAbs and PAbs reacted positively to microscopy negative urine samples in ELISA, however, there was a higher number of false positive reactivity by the PAbs than the MAbs. This observation may be explained as follows. MAbs are capable of recognizing and binding to only one specific antigenic determinant or epitope (Ansar and Ghosh, 2013) PAbs on the other hand, are capable of recognizing and binding different epitopes on one or different antigens with varying degree of affinity (Nakazawa et al., 2010). This higher antigen binding capacity of PAbs gives them and exaggerated reactivity to both specific and non-specific antigens, which results in the higher false positive rates that are characteristic of most PAb-based antigen detection assays (Nakazawa et al., 2010). In view of this, the higher reactivity and the corresponding higher false positive rates of the PAbs in microplate ELISA as compared to the MAbs, might be because they detected more Plasmodium negative urine samples non-specifically. Indeed, false positive results in malaria lead to non-judicious administration of anti- malarial drugs to people who do not really need them (Eibach et al., 2013; Azikiwe et al., 2012; Reyburn et al., 2007) and speeds up parasite development of resistant to such drugs (Eibach et al., 2013; Reyburn et al., 2007). Also, it causes delay in diagnosis and treatment of the actual infections responsible for the febrile signs and symptoms, which may lead to complications and death of the affected individuals (Reyburn et al., 2007). In a bid to arrest this diagnostic inaccuracy and its destructive outcomes, therefore, this study was designed to develop a highly sensitive and specific MAb urine-based dipstick test for diagnosis of malaria. The PAbs also reacted with some of the negative urine samples in ELISA but not in western blotting assay. This observation might be due to the fact that western blotting assay University of Ghana http://ugspace.ug.edu.gh 193 is more specific than microplate ELISA, which has a higher propensity to give false positive results (Bresnahan, 2013). Like the PAbs, the UCP4W7 MAbs also reacted with urine samples from both microscopy positive and negative individuals in ELISA, but not in western blotting assay. However, the lower false positive results in the UCP4W7 ELISA compared to the PAb ELISA might be because, the former was MAb-based, and could mostly detect only specific antigens with minimal cross-reactivity to non-target antigens. Also, the successful reactivity of the UCP4W7 MAb to protein bands in PAgHU, CPfAg and DUMIs but not with proteins in the negative control samples, measles and polio vaccines as well as the schistosome antigens, demonstrated the superior specificity of the MAb for malaria-related antigens. The differential detection of some protein bands in the profile of PAgHU by UCP4W7, which were not in CPfAg or DUMIs; and some protein bands in the DUMIs which were not present in PAgHU and CPfAg also suggested that perhaps, those proteins were not equally expressed in all the strains of parasites that infected the study participants. Indeed, this observation confirmed the reports made by Rosenthal (2012) and Gamboa et al. (2010). They reported that firstly, some P. falciparum parasites do not produce the parasite specific HRP2 because they lack the gene for expression of the antigen. Secondly, some expressed an altered HRP2 antigen. Lastly, some antigens like the HRP2 are not commonly expressed in the different species of the parasite. These characteristics are diagnostic limitations which lead to false negative results in some of the RDTs in some localities. False negative results, in turn, could lead to failure in treatment of individuals who have the disease. However, failure to treat children below 5 years who have acute P. falciparum infections for example, could lead to complicated malaria University of Ghana http://ugspace.ug.edu.gh 194 (Rosenthal, 2012). It is for these reasons that there is an urgent need to avoid false negative results in malaria diagnosis (Rosenthal, 2012). The limitations associated with the pre-existing RDTs provide interesting lessons to apply in the development of new diagnostic tools for malaria. These lessons suggested that perhaps, in the efforts to develop superior RDT-based tests for malaria, MAbs that have the capacity to detect pan-specific antigens in the urine of all infected individuals should be considered most. Also, they suggested that those proteins that were not common to all the samples could be explored for development of differential diagnostic tests required to make decisions on the type of antimalarial drugs to use. Out of the 3 vaccines and 2 antigens tested for cross-reactivity to the UCP4W7 MAb, yellow fever vaccine was observed to cross-react with the MAb. This observation suggested that probably, the urine of Plasmodium infected individuals used in generating the UCP4W7 MAb also had yellow fever vaccine or active viral antigens. Kim et al. (2010) and Goepp et al. (1992) have reported that vaccinated individuals do excrete vaccine antigens in their urine which cause false positive results in urine-based antigen detection assays. Also, Martinez et al. (2011) reported that yellow fever vaccine RNA or antigens could persist in vaccinated or infected individuals for more than 6 months as detected in their urine samples. These findings, therefore suggested that perhaps, the cross-reactivity observed between the UCP4W7 MAb and the yellow fever vaccine, was due to the induction of yellow fever binding affinity in the MAb by vaccine and/ or viral antigens which were probably present in the urine samples. This cross reactivity, however, was a limitation on the specificity of the UCP4W7 MAb for urinary Plasmodium antigens, which could lead to false positive results when used University of Ghana http://ugspace.ug.edu.gh 195 for diagnosis of malaria in suspected individuals. For these false positive results to be avoided therefore, yellow fever infections or vaccine antigens must be ruled out of such individuals. Furthermore, it is likely that further cloning of these hybridoma cells by limiting dilution to obtain a colony of cells arising from single parental cell lines, would yield second or third generation MAbs that are specific to only urinary Plasmodium antigens. Finally, the usefulness of the UCP4W7 MAb as a reagent for development of a urine- based diagnostic test for malaria was demonstrated by evaluating its sensitivity and specificity in microplate ELISA using microscopy as a gold standard test. The results showed that the relative sensitivity of the UCP4W7 ELISA was high (96.9%) although the relative specificity (75.6%) was not high enough. The rather low specificity of the UCP4W7 ELISA could be explained by the fact that microscopy was not specific enough which led to high false positive rates and a corresponding low specificity of the MAb ELISA. Several studies have shown that microscopy is not accurate enough to be used as a yardstick against which other tests should be evaluated (Steenkeste et al., 2010; Okell et al., 2009). Therefore, the findings of the study above, further emphasized the need for the use of more accurate tools as standards against which the performance of other tests would be assessed. Furthermore, the findings indicated that while the current prevalence of malaria and its destructive outcomes are still being battled with, perhaps, the development of additional tests to improve the diagnosis of the disease, would enhance current efforts to eradicate the disease as reported. In conclusion, this study provides useful information on the suitability of MAbs as diagnostic reagents for distinguishing between malaria infected and uninfected individuals. Also, it provides the base-line information that SDS-PAGE and/ or MAb-based western blotting assay could be explored in reliable semi quantitative techniques for differentiating between various levels of parasitaemia in infected individuals. Lastly, the study provides the University of Ghana http://ugspace.ug.edu.gh 196 evidence that urine is a reliable source of biomarker for development of non-invasive tests for diagnosis of malaria. Additions to Knowledge • MAb-secreting hybridoma cells have been produced against urinary Plasmodium antigens for the first time • A paper on the potential of urinary hepcidin as a promising diagnostic marker for malaria has been published • The research work has shown that SDS PAGE and WBA could be used to develop a urine-based semi-quantitative test for malaria • SDS-PAGE on infected human urine could be used to evaluate the efficacy of antimalarial drugs • Infected human urine may be a better alternative for assessment of antimalarial drug efficacy than blood Recommendations Further work, using PCR technology is required to confirm the true infection status of microscopy negative subjects. This is because, determination of the number of individuals who are truly negative is essential for ascertaining the specificity of the MAb-based test being developed. It is recommended that in further studies towards this end, more urine samples from individuals living outside malaria endemic regions would be used as true negative controls, compared with the microscopy determined negative control urine samples from the study community, to optimize the accuracy of the of the MAbs for detection of true positive infections. Also, additional work is required to produce single clones of the promising UCP4W7 MAb-secreting hybridoma cells for the purpose of obtaining MAbs for development and commercialization of the dipstick assay being developed. In addition, there is a need to investigate into the possibility of using membrane-based SDS-PAGE to ascertain malaria parasite density in infected and uninfected individuals. This University of Ghana http://ugspace.ug.edu.gh 197 is because, the success of this study could lead to the development of a quantitative or semi- quantitative urine-based test for malaria which is requisite for studies on anti-malaria drug efficacy. Furthermore, studies are required to investigate into the cross-reaction of the UCP4W7 with the yellow fever vaccine antigen. It is also highly desirable that further studies are conducted to determine the cross-reactivity of the UCP4W7 MAb with additional antigens from parasitic infections that share common clinical signs and symptoms with malaria which, however, were not examined in the studies reported in this thesis write-up. University of Ghana http://ugspace.ug.edu.gh 198 REFERENCES Ahmed, R. and D. Gray (1996). Immunological memory and protective immunity: Understanding their relation. Science 272, 54–60. Alemu, A., H.-P. Fuehrer, G. Getnet, A. Kassu, S. Getie and H. Noed (2014). 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University of Ghana http://ugspace.ug.edu.gh 224 APPENDICES APPENDIX 1: MAP OF KPONE-ON-SEA STUDY AREA Position On GT. Accra Regional Map Loc ation # # # # # KPONE TEMA ACCRA OLD NINGO PRAMPRAM C242 House Number Building Under Construction Foot Path Trunk Road Track Leg en d Com posed by CE RS GIS Univers ity of G hana Legon Tel 233 21 500301 Fax 233 21 500310 Nov. 20 04 Location Map # # # # # Loc ation KPONE TEMA ACCRA OLD NI GO PRAMPRAM N 1:6000 Scale 0.1 0 0.1 Kilometers 0.07 0 0.07 Miles K P O N E - O N - S E A #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S#S #S #S #S #S #S #S #S#S #S #S #S #S #S #S#S #S #S #S#S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S#S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S 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#S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S#S #S #S #S#S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S#S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S #S D08 4 D09 9 D08 3 D09 0 D16 9 D08 5 D08 5 D16 6 D16 8 D10 0 D08 6 D16 7 D16 5 D08 7 D11 3 D08 8 D20 0 D08 9 D10 6 D16 4 D10 9 D19 8 D16 3 D10 8 D06 9 D19 9 D16 2 D11 D11 2 D20 1 D07 3D10 7 D11 9 D11 0 D06 8 D16 0 D16 1 D11 8 D07 0 D15 7 A0 01 D06 7 D15 3 D15 9 D11 4 A0 02 D20 5 D15 6 D15 8 D15 5 D20 4 D20 7 D15 1 D15 4 D12 0 D21 0 D20 6 D15 3 D15 2 D20 8 A0 03 D12 1 D12 2 D11 7 A0 05 D13 1 D06 5 D11 6 A0 04 D06 4A0 07 D12 4 D14 6 D12 8 D14 7 A0 06 D06 0 D14 5 D12 7 D06 1 D12 5 A0 08 D06 2 D13 2 D05 8 D02 7 D13 0 D12 6 D02 6 D12 9 D06 3 D14 4 D05 6 D05 9 D13 3 D13 9 D13 4 D14 3 D14 8 A0 09 D13 6 D13 6 A0 10 A0 16 A0 17 PEN TECO ST CHUR CH D14 1 D05 0 D02 9 A0 11 D02 8 D02 0 D01 7 A0 12 D14 2 D01 8 D04 6 D04 8 A0 15 D05 5 D02 4 D01 6 D02 1 D02 5 A0 18 D04 7 A0 14 D04 9 BAR D01 5 D02 5 A0 14 A0 13 A0 14 A0 19 A0 67 A0 68 D05 4 D03 1 A0 69 A0 21 D01 3 A0 66 A0 49 D03 0D05 1 D01 4 A0 20 D03 2 DEEPER LI FE CHURC H D04 0 COM MU NIT Y CENT RE D05 2D03 9 D01 2 A0 65 A0 51 A0 45 D04 5A0 22 A0 46 D01 1 D05 3 A0 50 D04 1 D03 8 A0 23 A0 52 A0 61 D04 4 A0 43 D01 0 D04 2 BEST EVER SCH D03 6 D04 3 D00 2 A0 63 A0 47 A0 70 D00 1 A0 48 D03 7 A0 42 A0 62 A0 64 LAT RINE D00 9 A0 71 A0 72 D03 5 A2 04 A0 24 A0 41 A0 53 A0 73 D00 8 A0 44 A0 74 A2 04 A0 25 D03 4 A0 40 A0 34 A0 35 A0 55A0 39 A0 75 A0 38 D00 7 D03 3 SD A CHUR CHA0 32 A0 26 A0 77 A0 76 A0 80 A0 81 A0 30 K. V.I.P A0 79A0 29 A0 37 D00 4 A0 28 A1 39 A0 84 A1 36 A0 56 PR ESBY PRIM J.S.S A1 31 A1 32 D00 6 A1 35 KPO NE HEALTH CENTRE A0 27 A0 28 A1 34 A0 85 A0 86SC H BU SH (OL D C EMET ERY) FO OT BALL PARK D00 5K. V.I.P A1 25 A1 33 A1 28 A0 82 CORN MI LL A1 30 TO MB ST O NE A0 60 A1 26 A0 57 K. V.I.P SH ED(F ISH SMO KING ) A0 87 A1 29 A5 8 SC HOO L PRESBY CHUR CH A1 27 SC HOO L SC HOO L SC HOO L CORN MIL L GHANA TEL ECOM A0 59 A1 23 C21 9 C22 0 KPO NE PRESBY PRIM A A0 88 SC HOO L C18 1 C22 1 A0 89 KPO NE PRESBY PR IM B WAT ER T ANK A0 89 PR ESBY NURSERY A2 69 C22 2 C18 2 PO LI CE ST ATI ON C22 4 C22 3 C18 3C22 5 SC HOO L C18 9 K. V.I.P A2 71 PO ST OF F ICE A1 22A0 90 KPO NE TRAD IT ION COUN CIL SI LENT CAF E DIVINE CHURC H C18 4 C18 6 C18 8 A2 66 C17 9 A1 21 A0 92 DEVINE APO STOLI C CHURC H C18 7 C22 6 C18 5 A2 65 A2 67 A1 17 C24 3 A0 91 C19 5 A1 20 A2 64 C22 7 A1 17 A2 31 C18 9 A2 56 C24 2 C17 3 C17 8 A2 26C17 2 C19 1 A1 18A0 93A2 65 A0 96 SHED NET MENDING C17 1 C19 6 A2 55 C17 7 A2 27 A0 94 C22 8 C19 2 C19 4 A2 54 A2 28 A2 53 A1 15 DRINKING SPOT C17 6 C19 9 C19 8 A2 52 C17 4 A1 40 C29 9 A2 32 C19 7 A2 53 A2 59CORN MI LL A2 34 A2 25 A2 63 A0 95 C17 0 A2 51 A2 60 A2 58 C20 1 A2 37 C23 1 A2 30 C23 0 C20 0 CORN MI LL A2 62 A2 57 A0 98 C16 8 DANGME RURAL BANK A2 49 A2 35 A1 12 A2 61 C16 9 A2 50 A2 35 A2 24 C19 3 A2 22 A0 99 A2 48 LAT RINE A1 04 C21 7 A0 97 A2 47 C11 0 A0 97 A2 41 A2 05 C20 3 C23 2 C21 0 C21 8 C17 5 A2 38 C16 7 A1 41 C23 3 C21 4 A2 39 A2 06 A1 01A2 04 C20 4 A2 47 A1 06 C16 5 C23 4 A1 05 A1 11 A2 07 A1 13A2 03 C21 5 A2 01 C16 6 C16 4 C23 6 C02 7 A1 06 A2 19 A2 45 WT A2 12 A2 00 C20 5 C11 1 C15 9 C00 3 C20 9 C23 8 A1 03 A2 08 A1 02 A1 87 C20 7 C16 3 C16 1 C20 6 A2 02 A2 00 A1 10 C02 6 C00 2 A1 07 A1 42A1 85 C15 8 A1 99 C15 5 C00 1 C23 5 C10 9 C16 0 A2 11 C20 8 A1 87 FO OT BALL PARK C00 5 C01 1 A2 17 B0 27 CORN MI LL A1 86 C00 6 C03 0 C11 4 C01 0 C02 5 C15 0 C11 2 C02 9 A1 83 C23 7 C21 1 A1 08 C21 3 C02 4 A1 98C00 9 B0 24 A2 15C00 8 WT A1 43 C00 7 C14 7 C01 2 C02 3 A1 82 A1 96 C11 3 A1 88 C03 1 C01 3 C03 2 A1 81 SH RINE B0 25 A2 15 C03 4 A2 15 A2 14 B0 22 C03 3 A1 44 C15 7 C02 2 C02 0 B0 19 B0 28 C16 0 A1 50 C02 1 C15 6 C03 5 C14 8 A1 93 A1 90 A1 51 B0 21 C01 7 C16 2 C14 6 A1 89 C01 9 A1 45 A1 49 B0 29 B0 30C15 3 A1 52 C15 4 LAT RINE CAC CH URCH A1 95C11 5 A1 67 C14 5 B0 35 C03 9 C14 9 A1 48A1 92 C14 2C11 7 B0 33 C12 0 C15 1 B0 31 C15 2 A1 94 A1 46 C10 8 B0 32 B0 13 C14 0 B0 20 B0 18C10 4 B0 67 A1 92 B0 10 C04 1 B0 64 B0 66 B0 68 C10 7 A1 91 B0 71 C13 9 C03 8 B0 08 A1 47 C14 1 A1 55 C24 1 A1 66 B0 36 B0 72 A1 64 B0 41 B0 07 C13 8 C03 7 A1 63 B0 38 B0 42C10 6 B0 70 B0 73 B0 37 C04 2 B0 02 B0 78 C10 5 B0 39 C13 1 C10 1 B0 01 C12 2 B0 63 B0 06C13 7 C04 3 B0 45 A1 56 C13 0 C12 2 B0 65 B0 48 B0 05 A1 68 C03 6 C04 4 B0 46B0 77 C13 4 B0 47B0 76 B0 62 C02 5 C12 4 C13 2 A1 64 B0 61 B0 60 B0 59 C05 0 C13 3 B0 51 A1 59 C05 1 C09 9 C12 7 C04 5 B0 50 B0 74 C04 8 B1 64 B0 58 C12 8 B0 91 B0 86 B0 90 C10 2 B0 85 B0 84 PR EP SCH C05 3 C12 5 C04 6 C10 0 B0 55 C10 0 B0 87 C12 6 B0 93 C05 2 A1 60B0 89 C09 6 A1 74 B0 79 C09 8 C04 7 C05 3 B1 63 B0 88 B0 92 A1 73 B0 80 B0 54 C05 4 C09 2 B0 81 MET H CH URCH B0 56 B0 95 B0 53 C05 6 B0 57 B0 82 B1 07 C09 5 C09 7 B0 96 C09 0 C05 5 C08 3 C09 3 C08 8 B0 97 B1 62 C08 9 B0 94 C08 4 B1 06 B0 98 B1 61 C08 6 C08 2 B0 99 CORN MI LL B1 01 C08 7 B1 05 B1 00 C08 1 B1 60 B1 02 B1 08 B1 60 NIM TREES C10 3 B1 04 B1 59 B1 03 C23 9 C07 6 B1 12 C07 9 C05 7 C08 0 B1 56 C05 9 B1 19 B1 52 B1 18 B1 11 C05 8 B1 53 B1 13 B1 13B1 55 B1 09B1 13 B1 51C07 8 C06 0 C07 7 B1 50 B1 10 C06 1 B1 14NIM T REES C07 4 B1 47 B1 49 B1 64 B1 48 B1 15 C07 5 B1 64 B1 45 C24 0 B1 21 B1 44 C06 2 B1 17 B1 22B1 64APO STOLI C CHUR CH C07 3 B1 38 B1 64 B1 33 B1 23B1 40 C06 3 C06 6 B1 35 B1 37 C06 4 B1 36 C06 5 B1 32 C06 7 B1 35 B1 35 NUIT ED HEALIN G PRAYER GRO U B1 34 C07 0 B1 32 C06 8 B1 29 C06 8 C06 9 KPO NE CO M M PR EP SCH. B1 30 B1 29 B1 24 B1 25 C07 2 KPO NE CO M M. PRESBY SCH B1 26 C07 1 ST . MARY NURSERY SCH B1 27 A0 83 A0 78 A0 54 D12 3 D02 2 A2 70 A1 16 A1 14 A1 00 A1 84 A2 08 C22 4 C19 0 NIM T REES B1 58 B1 57 C21 6 A2 46 C02 8 C03 6 A1 61 B0 17 B0 15 B0 23 D14 0 D11 5 1282000 1282000 1283000 1283000 1284000 1284000 1285000 1285000 1286000 1286000 368000 368000 369000 369000 370000 370000 371000 371000 372000 372000 373000 373000 374000 374000 G U L F O F G U I N E A University of Ghana http://ugspace.ug.edu.gh 225 APPENDIX 2: ADDITIONAL DATA FROM CHAPTER FOUR Appendix 2a Variation of mean haemoglobin concentration among malaria positive subjects of same sex relative to age Appendix 2a summarizes the variation of the mean haemoglobin concentration (MHb) among malaria positive subjects of same sex with respect to age. According to these results all the female malaria positive subjects among the study community are anaemic, with a lower MHb than the normal value required for each category. The results also show that malaria positive female subjects above 15 years of age are the most severely anaemic (MHb = 10.605g/dL; normal MHb = 12 – 16 g/dL). Similarly, the male malaria positive subjects within the study community are anaemic, with MHb being lower than the normal range (children, MHb = 11 g/dL; adults, MHb = 14-18 g/dL). Comparably however, the male malaria positive subjects are more severely anaemic than their female counterparts. Generally, the 11 =15 year old positive male subjects are the most severely anaemic with a MHb being 2.6 g/dL lower than the normal range. 9.967 10.886 11.356 10.605 9.36 11.347 11.361 13.325 0 2 4 6 8 10 12 14 Under 6yrs 6 -10yrs 11 -15yrs Above15yrsM ea n H ae m o gl o b in C o u n tr at io n ( g/ d L) Age Positive Female(Hb) Positive Male(Hb) University of Ghana http://ugspace.ug.edu.gh 226 Appendix 2b Variation of malaria infection among study subjects relative to mean blood haemoglobin concentration and age Upon analyzing the variation of malaria infection among the study subjects with respect to MHb and age however, the results (Appendix 2b) show that generally, apart from the uninfected subjects under age 6 who have normal blood MHb (MHb = 11.083 g/dL), the rest of the subjects made up of infected subjects below 6 years of age through to both infected and uninfected subjects above 15 years old were anaemic with their MHb below the standard level for each age and sex category. Variation of Malaria Infection Among Study Subjects Relative to Mean Blood Haemoglobin Concentration and Age Appendix 2c Variation of mean haemoglobin concentration among malaria negative subjects of same sex relative to age Unlike the case for malaria positive subjects, the MHb is generally similar among male and female malaria negative subjects, and ranges from 11.05 g/dL in female negative subjects below 6 years of age to 11.61 g/dL in female negative subjects within the 11-15 years age category. In both the positive and negative cases however, the MHb (11.3 d/gL) of male 9.647 11.111 11.359 11.47611.083 11.324 11.559 11.623 0 2 4 6 8 10 12 14 Under 6yrs 6 -10yrs 11 -15yrs Above15yrs M e an H ae m o gl o b in c o n ce n tr at io n (g /d L) Age Positive Negati… University of Ghana http://ugspace.ug.edu.gh 227 subjects above 15 years old, even though it was lower than the standard range (14-18 g/dL) was not too far from the standard level. In totality, the two sets of results thus show that generally the MHb of both malaria positive and negative subjects within the study community follow a similar trend. Variation of Mean Blood Haemoglobin Concentration Among Malaria Negative Subjects of Same Sex with Respect to Age Appendix 2d Prevalence of Malaria Parasite Infection among Study Subjects as Determined by Commercial Rapid Diagnostic Test Appendix 2d summarizes the prevalence of malaria infection among the study subjects as determined by the commercial rapid diagnostic test kit. According to the graph, approximately 47.0% of the study participants has malaria while the simple majority were malaria negative. 11.111 11.446 11.497 13.349 11.052 11.256 11.611 11.044 0 2 4 6 8 10 12 14 16 Under 6yrs 6 -10yrs 11 -15yrs Above15yrs H ae m o gl o b in C o n tr at io n ( g/ d L) Age Negative Male(Hb) Negative Female(Hb) University of Ghana http://ugspace.ug.edu.gh 228 Prevalence of Malaria Parasite Infection among Study Subjects as Determined by Commercial Rapid Diagnostic Test Appendix 2e Prevalence of Anaemia among Study Subjects in Relation to Malaria Infection Determined by RDT Appendix 2e below, summarizes the prevalence of anaemia among the study subjects in relation to malaria infection as determined by the commercial rapid diagnostic test (RDT) kit. As shown, the prevalence of anaemia among malaria negative subjects (48.4%) is numerically higher than that (44.3%) among their malaria positive counterparts contrary to the normal expectation. Also even though the prevalence of anaemia among malaria negative subjects is numerically higher than that of malaria positive subjects, the difference is not statistically significant (p>0.05). 53.5 46.5 42 44 46 48 50 52 54 56 Negative Positive P re va le n ce o f M al ar ia ( % ) Infectivity Status University of Ghana http://ugspace.ug.edu.gh 229 Prevalence of Anaemia among Study Subjects in Relation to Malaria Infectivity as Determined by RDT 48.40% 44.30% 51.60% 55.70% 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% Negative Positive P re va le n ce o f A n ae m ia ( % ) Malaria Infectivity Status (χ2 = 2.18, p-value=0.140) Anaemic Non Anaemic University of Ghana http://ugspace.ug.edu.gh 230 APPENDIX 3: SCHEMATIC REPRESENTATION OF THE RAPID DIAGNOSTIC TEST PROCEDURE AS SHOWN IN WHO (2000) University of Ghana http://ugspace.ug.edu.gh 231 APPENDIX 4: RESULTS OF URINE ANALYSIS Test Characteristic/Parameter Test Results Frequency Percent Leukocytes (WBC/µL) Neg 1095 91.4 ± 9 .8 + 64 5.3 ++ 17 1.4 +++ 13 1.1 Total 1198 100.0 Nitrite Neg 1203 98.0 Pos 24 2.0 Total 1227 100.0 Urobilinogen (mg/dl; µmol/L) Neg 1117 90.2 1 [17] 60 4.8 2 [35] 43 3.5 4 [70] 11 .9 8 [140] 7 .6 Total 1238 100.0 Protein (mg/dl; (g/L) Neg 781 69.4 ± [30; (0.15)] 43 3.8 + [30; (0.3)] 234 20.8 ++ [100; (1.0)] 52 4.6 +++ [300; (3.0)] 12 1.1 ++++ [2000; (20)] 3 .3 Total 1125 100.0 pH 5.0 491 40.3 6.0 547 44.9 7.0 94 7.7 8.0 75 6.2 9.0 12 1.0 Total 1219 100.0 BLOOD (RBC/µL) Neg 1115 90.1 ± 2 .2 + 46 3.7 ++ 29 2.3 +++ 32 2.6 5 - 10(1) 8 .6 5 - 10 6 .5 Total 1238 100.0 Specific Gravity (SG) None 8 .6 1.000 427 34.6 1.005 52 4.2 1.010 146 11.8 1.015 146 11.8 1.020 207 16.8 1.025 144 11.7 University of Ghana http://ugspace.ug.edu.gh 232 1.030 104 8.4 Total 1234 100.0 etones (mg/dl; mmol/L) Neg 1065 86.4 ± [ 5 (0.5)] 54 4.4 15 (1.5) 43 3.5 40 (4.0) 11 .9 80 (8.0) 43 3.5 160 (16) 16 1.3 Total 1232 100.0 Bilirubin (mg/dl; mmol/L) Neg 1207 98.0 + [ 1(17) ] 20 1.6 ++ [ 2(35) ] 5 .4 Total 1232 100.0 Glucose (mg/dl; mmol/L) Neg 1216 98.5 100(5) 10 .8 250(15) 5 .4 500(30) 2 .2 1000(60) 2 .2 Total 1235 100.0 Ascorbic Acid (mg/dl; mmol/L) Neg 416 84.4 + [ 10(5.6) ] 65 13.2 ++ [ 20(1.14) ] 12 2.4 Total 493 100.0 University of Ghana http://ugspace.ug.edu.gh 233 Urine Analysis with Respect to Age (Positive Cases) Under 6yrs 6 -10yrs 11 -15yrs Above 15yrs Count Row N % Count Row N % Count Row N % Count Row N % Leukocytes (WBC/µL) Neg 98 18.8% 140 26.9% 81 15.5% 202 38.8% ± 0 0.0% 2 50.0% 1 25.0% 1 25.0% + 6 30.0% 6 30.0% 2 10.0% 6 30.0% ++ 0 0.0% 0 0.0% 1 25.0% 3 75.0% +++ 0 0.0% 0 0.0% 1 16.7% 5 83.3% Nitrite Neg 103 18.4% 151 27.0% 89 15.9% 217 38.8% Pos 2 16.7% 3 25.0% 0 0.0% 7 58.3% Urobilinogen (mg/dl; µmol/L) Neg 89 18.1% 142 28.9% 74 15.0% 187 38.0% 1 [17] 4 10.8% 8 21.6% 5 13.5% 20 54.1% 2 [35] 11 35.5% 3 9.7% 5 16.1% 12 38.7% 4 [70] 0 0.0% 0 0.0% 4 44.4% 5 55.6% 8 [140] 2 33.3% 1 16.7% 1 16.7% 2 33.3% 12 [200] 0 0.0% 0 0.0% 0 0.0% 0 0.0% 0.2 [3.5] 0 0.0% 0 0.0% 0 0.0% 0 0.0% Protein (mg/dl; (g/L) Neg 55 17.8% 88 28.5% 50 16.2% 116 37.5% ± [30; (0.15)] 5 17.2% 9 31.0% 7 24.1% 8 27.6% + [30; (0.3)] 25 18.0% 35 25.2% 22 15.8% 57 41.0% ++ [100; (1.0)] 11 27.5% 11 27.5% 4 10.0% 14 35.0% +++ [300; (3.0)] 3 30.0% 1 10.0% 1 10.0% 5 50.0% ++++ [2000; (20)] 0 0.0% 0 0.0% 0 0.0% 2 100.0% pH Mean 5.7 5.6 5.9 University of Ghana http://ugspace.ug.edu.gh 234 BLOOD (RBC/µL) Neg 96 18.3% 148 28.2% 82 15.6% 198 37.8% ± 0 0.0% 0 0.0% 0 0.0% 0 0.0% + 6 27.3% 3 13.6% 1 4.5% 12 54.5% ++ 1 6.7% 1 6.7% 6 40.0% 7 46.7% +++ 3 25.0% 2 16.7% 0 0.0% 7 58.3% 5 - 10(1) 0 0.0% 0 0.0% 0 0.0% 2 100.0% 5 - 10 0 0.0% 0 0.0% 0 0.0% 0 0.0% Specific Gravity (SG) None 0 0.0% 1 25.0% 0 0.0% 3 75.0% 1.000 40 20.5% 37 19.0% 25 12.8% 93 47.7% 1.005 8 40.0% 4 20.0% 2 10.0% 6 30.0% 1.010 12 19.7% 20 32.8% 13 21.3% 16 26.2% 1.015 9 12.5% 23 31.9% 10 13.9% 30 41.7% 1.020 18 16.5% 30 27.5% 21 19.3% 40 36.7% 1.025 11 15.7% 23 32.9% 12 17.1% 24 34.3% 1.030 7 17.1% 15 36.6% 6 14.6% 13 31.7% Ketones (mg/dl; mmol/L) Neg 71 15.1% 112 23.9% 77 16.4% 209 44.6% ± [ 5 (0.5)] 8 23.5% 12 35.3% 5 14.7% 9 26.5% 15 (1.5) 9 37.5% 7 29.2% 4 16.7% 4 16.7% 40 (4.0) 4 66.7% 2 33.3% 0 0.0% 0 0.0% 80 (8.0) 12 41.4% 14 48.3% 2 6.9% 1 3.4% 160 (16) 1 10.0% 6 60.0% 1 10.0% 2 20.0% Bilirubin (mg/dl; mmol/L) Neg 102 18.5% 147 26.6% 87 15.8% 216 39.1% + [ 1(17) ] 2 14.3% 4 28.6% 2 14.3% 6 42.9% ++ [ 2(35) ] 0 0.0% 2 50.0% 0 0.0% 2 50.0% +++ [ 4(70) ] 0 0.0% 0 0.0% 0 0.0% 0 0.0% University of Ghana http://ugspace.ug.edu.gh 235 Glucose (mg/dl; mmol/L) Neg 103 18.4% 152 27.1% 86 15.4% 219 39.1% 100(5) 1 16.7% 1 16.7% 1 16.7% 3 50.0% 250(15) 0 0.0% 0 0.0% 1 25.0% 3 75.0% 500(30) 1 50.0% 0 0.0% 1 50.0% 0 0.0% 1000(60) 0 0.0% 0 0.0% 0 0.0% 0 0.0% ≥2000(110) 0 0.0% 0 0.0% 0 0.0% 0 0.0% Ascorbic Acid (mg/dl; mmol/L) Neg 35 21.0% 41 24.6% 23 13.8% 68 40.7% + [ 10(5.6) ] 5 18.5% 10 37.0% 5 18.5% 7 25.9% ++ [ 20(1.14) ] 0 0.0% 3 60.0% 1 20.0% 1 20.0% +++ [ 40(2.28) ] 0 0.0% 0 0.0% 0 0.0% 0 0.0% Urine Analysis with Respect to Age Negative Cases Under 6yrs 6 -10yrs 11 -15yrs Above15yrs Count Row N % Count Row N % Count Row N % Count Row N % Leukocytes (WBC/µL) Neg 96 16.7% 88 15.3% 58 10.1% 332 57.8% ± 0 0.0% 0 0.0% 1 20.0% 4 80.0% + 4 9.1% 5 11.4% 2 4.5% 33 75.0% ++ 0 0.0% 2 15.4% 0 0.0% 11 84.6% +++ 0 0.0% 1 14.3% 1 14.3% 5 71.4% Nitrite Neg 99 15.4% 99 15.4% 65 10.1% 380 59.1% Pos 2 16.7% 1 8.3% 1 8.3% 8 66.7% University of Ghana http://ugspace.ug.edu.gh 236 Urobilinogen (mg/dl; µmol/L) Neg 99 15.8% 95 15.2% 63 10.1% 368 58.9% 1 [17] 2 8.7% 3 13.0% 4 17.4% 14 60.9% 2 [35] 0 0.0% 0 0.0% 1 8.3% 11 91.7% 4 [70] 0 0.0% 2 100.0% 0 0.0% 0 0.0% 8 [140] 0 0.0% 0 0.0% 0 0.0% 1 100.0% 12 [200] 0 0.0% 0 0.0% 0 0.0% 0 0.0% 0.2 [3.5] 0 0.0% 0 0.0% 0 0.0% 0 0.0% Protein (mg/dl; (g/L) Neg 83 17.6% 78 16.5% 49 10.4% 262 55.5% ± [30; (0.15)] 1 7.1% 5 35.7% 4 28.6% 4 28.6% + [30; (0.3)] 9 9.5% 12 12.6% 10 10.5% 64 67.4% 2+ [100; (1.0)] 0 0.0% 0 0.0% 0 0.0% 12 100.0% 3+ [300; (3.0)] 0 0.0% 0 0.0% 0 0.0% 2 100.0% 4+ [2000; (20)] 0 0.0% 0 0.0% 0 0.0% 1 100.0% pH Mean 5.7 5.8 5.8 6.0 BLOOD (RBC/µL) Neg 99 16.8% 98 16.6% 63 10.7% 331 56.0% ± 0 0.0% 0 0.0% 1 50.0% 1 50.0% + 2 8.3% 2 8.3% 1 4.2% 19 79.2% ++ 0 0.0% 0 0.0% 1 7.1% 13 92.9% +++ 0 0.0% 0 0.0% 1 5.0% 19 95.0% 5 - 10(1) 0 0.0% 0 0.0% 0 0.0% 6 100.0% 5 - 10 0 0.0% 0 0.0% 1 16.7% 5 83.3% Specific Gravity (SG) None 3 75.0% 0 0.0% 0 0.0% 1 25.0% 1.000 31 13.4% 28 12.1% 18 7.8% 155 66.8% 1.005 6 18.8% 5 15.6% 3 9.4% 18 56.2% 1.010 17 20.0% 15 17.6% 10 11.8% 43 50.6% 1.015 7 9.5% 15 20.3% 11 14.9% 41 55.4% 1.020 20 20.4% 13 13.3% 13 13.3% 52 53.1% 1.025 5 6.8% 12 16.2% 8 10.8% 49 66.2% 1.030 12 19.0% 12 19.0% 4 6.3% 35 55.6% Neg 77 12.9% 85 14.3% 61 10.2% 373 62.6% University of Ghana http://ugspace.ug.edu.gh 237 Ketones (mg/dl; mmol/L) ± [ 5 (0.5)] 5 25.0% 4 20.0% 3 15.0% 8 40.0% 15 (1.5) 7 36.8% 3 15.8% 1 5.3% 8 42.1% 40 (4.0) 3 60.0% 1 20.0% 1 20.0% 0 0.0% 80 (8.0) 4 28.6% 6 42.9% 2 14.3% 2 14.3% 160 (16) 4 66.7% 1 16.7% 0 0.0% 1 16.7% Bilirubin (mg/dl; mmol/L) Neg 99 15.1% 100 15.3% 67 10.2% 389 59.4% + [ 1(17) ] 2 33.3% 0 0.0% 1 16.7% 3 50.0% ++ [ 2(35) ] 0 0.0% 0 0.0% 0 0.0% 1 100.0% +++ [ 4(70) ] 0 0.0% 0 0.0% 0 0.0% 0 0.0% Glucose (mg/dl; mmol/L) Neg 98 14.9% 100 15.2% 68 10.4% 390 59.5% 100(5) 2 50.0% 0 0.0% 0 0.0% 2 50.0% 250(15) 1 100.0% 0 0.0% 0 0.0% 0 0.0% 500(30) 0 0.0% 0 0.0% 0 0.0% 0 0.0% 1000(60) 0 0.0% 0 0.0% 0 0.0% 2 100.0% ≥ 2000(110) 0 0.0% 0 0.0% 0 0.0% 0 0.0% Ascorbic Acid (mg/dl; mmol/L) Neg 48 19.3% 35 14.1% 22 8.8% 144 57.8% + [ 10(5.6) ] 4 10.5% 9 23.7% 5 13.2% 20 52.6% ++ [20(1.14)] 0 0.0% 2 28.6% 0 0.0% 5 71.4% +++ [40(2.28) ] 0 0.0% 0 0.0% 0 0.0% 0 0.0% University of Ghana http://ugspace.ug.edu.gh 238 Patient Urine Test Analysis Test against Microscopy Test and ICT Strip Parameter Multistix 10 SG Reagent Strip Microscopy ICT Strip Positivity Grade (Level of Parasitaemia) No MPS 1+ [40 - 4999] 2+ [5000 - 49999] 3+ [50000 - 99999] 4+ [ >100000] Negative Positive Leukocytes (WBC/µL) Neg 52.4% 26.0% 12.1% 4.4% 5.1% 52.4% 47.6% ± 55.6% 22.2% 11.1% 11.1% 0.0% 55.6% 44.4% + 68.8% 25.0% 4.7% 1.6% 0.0% 70.3% 29.7% ++ 76.5% 23.5% 0.0% 0.0% 0.0% 76.5% 23.5% +++ 53.8% 46.2% 0.0% 0.0% 0.0% 46.2% 53.8% Nitrite Neg 53.4% 25.7% 12.1% 4.2% 4.7% 53.6% 46.4% Pos 50.0% 33.3% 8.3% 0.0% 8.3% 45.8% 54.2% Urobilinogen (mg/dl; µmol/L) Neg 56.0% 25.1% 10.7% 3.8% 4.5% 54.9% 45.1% 1 [17] 38.3% 31.7% 21.7% 3.3% 5.0% 41.7% 58.3% 2 [35] 27.9% 34.9% 16.3% 11.6% 9.3% 51.2% 48.8% 4 [70] 18.2% 27.3% 36.4% 9.1% 9.1% 27.3% 72.7% 8 [140] 14.3% 28.6% 42.9% 0.0% 14.3% 14.3% 85.7% 12 [200] 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.2 [3.5] 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Protein (mg/dl; (g/L) Neg 60.4% 23.3% 9.9% 3.5% 2.9% 58.4% 41.6% ± [30; (0.15)] 32.6% 48.8% 9.3% 2.3% 7.0% 23.3% 76.7% + [30; (0.3)] 40.6% 26.1% 20.5% 5.6% 7.3% 41.0% 59.0% ++ [100; (1.0)] 23.1% 26.9% 21.2% 13.5% 15.4% 23.1% 76.9% +++ [300; (3.0)] 16.7% 66.7% 0.0% 8.3% 8.3% 16.7% 83.3% ++++ [2000; (20)] 33.3% 66.7% 0.0% 0.0% 0.0% 33.3% 66.7% University of Ghana http://ugspace.ug.edu.gh 239 pH Mean 5.9 5.8 5.7 5.5 5.6 5.9 5.7 BLOOD (RBC/µL) Neg 53.0% 26.2% 12.0% 4.1% 4.7% 52.4% 47.6% ± 100.0% 0.0% 0.0% 0.0% 0.0% 100.0% 0.0% + 52.2% 23.9% 13.0% 0.0% 10.9% 63.0% 37.0% ++ 48.3% 31.0% 17.2% 3.4% 0.0% 58.6% 41.4% +++ 62.5% 18.8% 6.2% 6.2% 6.2% 62.5% 37.5% 5 - 10(1) 75.0% 12.5% 0.0% 12.5% 0.0% 75.0% 25.0% 5 - 10 100.0% 0.0% 0.0% 0.0% 0.0% 100.0% 0.0% Specific Gravity (SG) None 50.0% 50.0% 0.0% 0.0% 0.0% 50.0% 50.0% 1.000 54.3% 26.5% 12.2% 2.1% 4.9% 67.9% 32.1% 1.005 61.5% 30.8% 3.8% 3.8% 0.0% 53.8% 46.2% 1.010 58.2% 23.3% 11.0% 3.4% 4.1% 47.9% 52.1% 1.015 50.7% 32.2% 8.9% 4.8% 3.4% 42.5% 57.5% 1.020 47.3% 21.7% 17.4% 7.2% 6.3% 40.6% 59.4% 1.025 51.4% 24.3% 12.5% 6.9% 4.9% 46.5% 53.5% 1.030 60.6% 23.1% 9.6% 1.9% 4.8% 54.8% 45.2% Ketones (mg/dl; mmol/L) Neg 56.0% 25.7% 11.0% 3.7% 3.7% 55.6% 44.4% ± [ 5 (0.5)] 37.0% 33.3% 16.7% 7.4% 5.6% 44.4% 55.6% 15 (1.5) 44.2% 23.3% 16.3% 2.3% 14.0% 46.5% 53.5% 40 (4.0) 45.5% 0.0% 0.0% 27.3% 27.3% 45.5% 54.5% 80 (8.0) 32.6% 23.3% 27.9% 7.0% 9.3% 32.6% 67.4% 160 (16) 37.5% 37.5% 12.5% 0.0% 12.5% 31.2% 68.8% Bilirubin (mg/dl; mmol/L) Neg 54.3% 25.4% 11.8% 4.0% 4.5% 53.8% 46.2% + [ 1(17) ] 30.0% 30.0% 20.0% 5.0% 15.0% 50.0% 50.0% ++ [ 2(35) ] 20.0% 60.0% 0.0% 20.0% 0.0% 20.0% 80.0% +++ [ 4(70) ] 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Glucose Neg 53.9% 25.7% 11.9% 3.9% 4.5% 53.8% 46.2% University of Ghana http://ugspace.ug.edu.gh 240 (mg/dl; mmol/L) 100(5) 40.0% 30.0% 10.0% 0.0% 20.0% 60.0% 40.0% 250(15) 20.0% 40.0% 20.0% 20.0% 0.0% 40.0% 60.0% 500(30) 0.0% 50.0% 0.0% 50.0% 0.0% 0.0% 100.0% 1000(60) 100.0% 0.0% 0.0% 0.0% 0.0% 50.0% 50.0% ≥2000(110) 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Ascorbic Acid (mg/dl; mmol/L) Neg 59.9% 22.4% 7.9% 5.8% 4.1% 50.7% 49.3% + [ 10(5.6) ] 58.5% 15.4% 10.8% 6.2% 9.2% 43.1% 56.9% ++ [ 20(1.14) ] 58.3% 8.3% 16.7% 0.0% 16.7% 58.3% 41.7% +++ [ 40(2.28)] 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% University of Ghana http://ugspace.ug.edu.gh 241 Urine Test Analysis Test against Microscopy Test and ICT Strip Malaria POSITIVE Cases Only Parameter Multistix SG Reagent Strip Microscopy ICT Strip Positivity Grade (Level of Parasitaemia) 1+ [40 - 4999] 2+ [5000 - 49999] 3+ [50000 - 99999] 4+ [ >100000] Negative Positive Leukocytes (WBC/µL) Neg 54.7% 25.3% 9.2% 10.7% 13.4% 86.6% ± 50.0% 25.0% 25.0% 0.0% 0.0% 100.0% + 80.0% 15.0% 5.0% 0.0% 10.0% 90.0% ++ 100.0% 0.0% 0.0% 0.0% 0.0% 100.0% +++ 100.0% 0.0% 0.0% 0.0% 0.0% 100.0% Nitrite Neg 55.2% 25.9% 8.9% 10.0% 12.9% 87.1% Pos 66.7% 16.7% 0.0% 16.7% 0.0% 100.0% Urobilinogen (mg/dl; µmol/L) Neg 56.9% 24.4% 8.5% 10.2% 10.8% 89.2% 1 [17] 51.4% 35.1% 5.4% 8.1% 21.6% 78.4% 2 [35] 48.4% 22.6% 16.1% 12.9% 35.5% 64.5% 4 [70] 33.3% 44.4% 11.1% 11.1% 11.1% 88.9% 8 [140] 33.3% 50.0% 0.0% 16.7% 0.0% 100.0% 12 [200] 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.2 [3.5] 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Protein (mg/dl; (g/L) Neg 58.9% 24.9% 8.7% 7.4% 11.3% 88.7% ± [30; (0.15)] 72.4% 13.8% 3.4% 10.3% 0.0% 100.0% + [30; (0.3)] 43.9% 34.5% 9.4% 12.2% 11.5% 88.5% ++ [100; (1.0)] 35.0% 27.5% 17.5% 20.0% 5.0% 95.0% +++ [300; (3.0)] 80.0% 0.0% 10.0% 10.0% 0.0% 100.0% ++++ [2000; (20)] 100.0% 0.0% 0.0% 0.0% 0.0% 100.0% pH Mean 5.8 5.7 5.5 5.6 6.2 5.7 BLOOD (RBC/µL) Neg 55.7% 25.6% 8.8% 9.9% 11.1% 88.9% ± 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% University of Ghana http://ugspace.ug.edu.gh 242 + 50.0% 27.3% 0.0% 22.7% 40.9% 59.1% ++ 60.0% 33.3% 6.7% 0.0% 26.7% 73.3% +++ 50.0% 16.7% 16.7% 16.7% 16.7% 83.3% 5 - 10(1) 50.0% 0.0% 50.0% 0.0% 0.0% 100.0% 5 - 10 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Specific Gravity (SG) None 100.0% 0.0% 0.0% 0.0% 0.0% 100.0% 1.000 57.9% 26.7% 4.6% 10.8% 32.8% 67.2% 1.005 80.0% 10.0% 10.0% 0.0% 0.0% 100.0% 1.010 55.7% 26.2% 8.2% 9.8% 1.6% 98.4% 1.015 65.3% 18.1% 9.7% 6.9% 1.4% 98.6% 1.020 41.3% 33.0% 13.8% 11.9% 0.9% 99.1% 1.025 50.0% 25.7% 14.3% 10.0% 5.7% 94.3% 1.030 58.5% 24.4% 4.9% 12.2% 2.4% 97.6% Ketones (mg/dl; mmol/L) Neg 58.4% 24.9% 8.3% 8.3% 13.4% 86.6% ± [ 5 (0.5)] 52.9% 26.5% 11.8% 8.8% 17.6% 82.4% 15 (1.5) 41.7% 29.2% 4.2% 25.0% 8.3% 91.7% 40 (4.0) 0.0% 0.0% 50.0% 50.0% 16.7% 83.3% 80 (8.0) 34.5% 41.4% 10.3% 13.8% 0.0% 100.0% 160 (16) 60.0% 20.0% 0.0% 20.0% 0.0% 100.0% Bilirubin (mg/dl; mmol/L) Neg 55.6% 25.9% 8.7% 9.8% 12.0% 88.0% + [ 1(17) ] 42.9% 28.6% 7.1% 21.4% 28.6% 71.4% ++ [ 2(35) ] 75.0% 0.0% 25.0% 0.0% 0.0% 100.0% +++ [ 4(70) ] 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Glucose (mg/dl; mmol/L) Neg 55.7% 25.9% 8.6% 9.8% 12.3% 87.7% 100(5) 50.0% 16.7% 0.0% 33.3% 33.3% 66.7% 250(15) 50.0% 25.0% 25.0% 0.0% 25.0% 75.0% 500(30) 50.0% 0.0% 50.0% 0.0% 0.0% 100.0% 1000(60) 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% University of Ghana http://ugspace.ug.edu.gh 243 ≥2000(110) 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Ascorbic Acid (mg/dl; mmol/L) Neg 55.7% 19.8% 14.4% 10.2% 0.0% 100.0% + [ 10(5.6) ] 37.0% 25.9% 14.8% 22.2% 0.0% 100.0% ++ [ 20(1.14) ] 20.0% 40.0% 0.0% 40.0% 0.0% 100.0% +++ [ 40(2.28) ] 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% University of Ghana http://ugspace.ug.edu.gh 244 Urine Test Analysis Test against Microscopy Test and ICT Strip Malaria Negative Cases Only MPS Grade ICT Strip Parameter Test Results No MPS Negative Positive Leukocytes (WBC/µL) Neg 100.0% 88.0% 12.0% ± 100.0% 100.0% 0.0% + 100.0% 97.7% 2.3% ++ 100.0% 100.0% 0.0% +++ 100.0% 85.7% 14.3% Nitrite Neg 100.0% 89.1% 10.9% Pos 100.0% 91.7% 8.3% Urobilinogen (mg/dl; µmol/L) Neg 100.0% 89.6% 10.4% 1 [17] 100.0% 73.9% 26.1% 2 [35] 100.0% 91.7% 8.3% 4 [70] 100.0% 100.0% 0.0% 8 [140] 100.0% 100.0% 0.0% 12 [200] 100.0% 0.0% 0.0% 0.2 [3.5] 100.0% 0.0% 0.0% Protein (mg/dl; (g/L) Neg 100.0% 89.2% 10.8% ± [30; (0.15)] 100.0% 71.4% 28.6% + [30; (0.3)] 100.0% 84.2% 15.8% ++ [100; (1.0)] 100.0% 83.3% 16.7% +++ [300; (3.0)] 100.0% 100.0% 0.0% ++++ [2000; (20)] 100.0% 100.0% 0.0% University of Ghana http://ugspace.ug.edu.gh 245 pH 5.9 BLOOD (RBC/µL) Neg 100.0% 89.0% 11.0% ± 100.0% 100.0% 0.0% + 100.0% 83.3% 16.7% ++ 100.0% 92.9% 7.1% +++ 100.0% 90.0% 10.0% 5 - 10(1) 100.0% 100.0% 0.0% 5 - 10 100.0% 100.0% 0.0% Specific Gravity (SG) None 100.0% 100.0% 0.0% 1.000 100.0% 97.4% 2.6% 1.005 100.0% 87.5% 12.5% 1.010 100.0% 81.2% 18.8% 1.015 100.0% 82.4% 17.6% 1.020 100.0% 84.7% 15.3% 1.025 100.0% 85.1% 14.9% 1.030 100.0% 88.9% 11.1% Ketones (mg/dl; mmol/L) Neg 100.0% 88.8% 11.2% ± [ 5 (0.5)] 100.0% 90.0% 10.0% 15 (1.5) 100.0% 94.7% 5.3% 40 (4.0) 100.0% 80.0% 20.0% 80 (8.0) 100.0% 100.0% 0.0% 160 (16) 100.0% 83.3% 16.7% Bilirubin (mg/dl; mmol/L) Neg 100.0% 89.0% 11.0% + [ 1(17) ] 100.0% 100.0% 0.0% ++ [ 2(35) ] 100.0% 100.0% 0.0% University of Ghana http://ugspace.ug.edu.gh 246 +++ [ 4(70) ] 100.0% Glucose (mg/dl; mmol/L) Neg 100.0% 89.2% 10.8% 100(5) 100.0% 100.0% 0.0% 250(15) 100.0% 100.0% 0.0% 500(30) 0.0% 0.0% 0.0% 1000(60) 100.0% 50.0% 50.0% ≥2000(110) 0.0% 0.0% 0.0% Ascorbic Acid (mg/dl; mmol/L) Neg 100.0% 84.7% 15.3% + [ 10(5.6) ] 100.0% 73.7% 26.3% ++ [ 20(1.14) ] 100.0% 100.0% 0.0% +++ [ 40(2.28) ] 0.0% 0.0% 0.0% University of Ghana http://ugspace.ug.edu.gh 247 APPENDIX 5: PUBLICATIONS Appendix 5a A poster presented at the First Annual College of Health Sciences Conference, held on 26th to 28th September, 2007 Introduction, Background and Rationale Rapid, safe, simple and accurate diagnosis is required for effective management and control of malaria (2), however, the major current diagnostic tools (clinical, blood smear microscopy and the immuno-chromatographic tests) lack these qualities. These limitations that beset diagnosis of malaria make management and control of the disease very difficult. This study is therefore being carried out to develop a simple, accurate, non-invasive and rapid urine-based dipstick assay (UBRDT) for alternative diagnosis of malaria. Patients, Materials and Methods Study Site: The study field site is Kpone On Sea, a coastal fishing village in south eastern Ghana. Study Subjects: Patients visiting Kpone health centre and pregnant women on antenatal care. Methodology: Plasmodium parasite infectivity and anaemia were determined by Blood-Smear Microscopy and the Quantitative Buffy Coat (QBC) test. Urinary malaria proteins for UBRDT development were identified and analyzed by one-dimensional SDS-PAGE, Western Blot Analysis, Mass Spectrometry Sequencing (MSS) and a urine-based malaria antigen-detection ELISA (UBMADE) being evaluated. Plasmodium antigens extracted from urine samples were used to immunize Balb/C mice towards monoclonal antibody (Moab) production and UBRDT development. Results Microscopy of blood samples from 290 recruited patients showed that 151 (52.1%) were malaria positive (Table 1). This included 24/58 (41.4%) of pregnant women (Table 3). In all 23/290 (7.9%) had severe malaria with parasitaemia >100,000 parasites/μl blood and 82 (28.3%) had mild malaria with <5,000 parasites/μl blood (Table 2). Generally, the parasitaemia ranged from 40 - 394,480 (Median = 3,520 parasites/ μl blood). The UBMADE and Western blot analysis detected malaria urinary antigens in 53 (35.1%) of the 151 malaria positive patients. Of the 151 malaria positive individuals, 43 (28.5%) were febrile and 108 afebrile. Also, 26/43 (60.5%) of the febrile individuals were antigen positive, whilst only (25.0%) of the 108 afebrile cases had the malaria urinary antigen. The geometric mean parasitaemia in febrile versus afebrile groups was 8,109:1320. In the SDS-PAGE/Western Blot analyses, a P. chabaudi infected mouse serum detected a 30- 35kDa band that was present in both purified P. falciparum antigen and malaria positive urine. This protein is undergoing MSS for identification. Production of anti-urinary-Plasmodium Moabs for development of the UBRDT is also underway. Discussions and Comments The observation that a high proportion (41.4%) of the asymptomatic pregnant women (Table 3) attending antenatal clinic were infected with Plasmodium parasites may raise questions about the effectiveness of intermittent preventive treatment (IPT), which could be influenced by compliance, and drug failure even though this is less likely. This observation therefore calls for further research to determine the reasons for high malaria prevalence among pregnant women; and reaffirms the importance of preventive treatment in pregnancy. Generally, children aged 5 years and below who are reported to be more susceptible (3) to the disease had the highest proportion 40/57 (70.2%) of malaria cases (Table 3), of which 4/40 (10%) arrived at the health centre either comatose or convulsive, indicating the need for early diagnosis and treatment. Even though it has been reported that malaria causes elevation in the amounts of proteins, bilirubin and blood excreted in urine (1), it is difficult to infer from this study (Table 1). Additional data are therefore required to make any conclusive deduction. Findings from this study show that a majority of the individuals from the study community are anaemic (Table 2). However, there is no statistical difference between the Plasmodium-infected anaemic and the uninfected anaemic groups (p>0.05). Conclusion In view of the observed prevalence of Plasmodium infections in pregnant women and children <5 years who were anaemic, the development of a rapid and accurate diagnostic tool for malaria would promote timely detection of infections in vulnerable risk groups. Studies Towards the Development of an Accurate Urine-Based Rapid Diagnostic Test (UBRDT) for Malaria McKakpo U.S., K.M. Bosompem, E.A.K. Addison, D. Sullivan Jnr., P. Amankwah, I. Ankrah, P. Madjitey, F. Lomotey, N. Amoah, A.N. Noye, M. Mensah, W.A. Lomotey and I.A. Quakyi Acknowledgements School of Public Health, College of Health Sciences, University of Ghana, Legon. The Department of Population, Family and Reproductive Health, School of Public Health, University of Ghana, Legon. Gates Institute for Population and Reproductive Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA. Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA. Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon. MIM/TDR/WHO. The Kpone Health Centre. The staff of the above named institutions. The Kpone study community References 1. Rodriguez-del Valle, M. et al. (1991. Journal of Clinical Microbiology, 29 (6): 1236-1242. 2. WHO (1993). A global strategy for malaria control, Geneva. 3. WHO/OMS (2002). Malaria. WHO Fact Sheet No 94. Table 2. Relationship between clinical outcomes of malaria, parasitaemia and anaemia Clinical Outcome Number of Subjects Parasites per microlitre of Blood Number Anaemic Severe 23 >100,000 23 Moderate 46 99,999-5000 45 Asymptomatic 82 4,999-40 68 Uninfected 139 0 117 Total 151 - 253 Table 3. Plasmodium infection rates in different risk groups among study subjects Malaria Positive Subjects Number Tested No. (%) Pregnant Women 58 24 (41.4) 5 years and below 57 40 (70.2) > 5 years 175 87 (49.7) Total 290 151 (52.1) Table 1. Relative sensitivity and specificity of various tests in detecting Plasmodium parasite infection Test Number Tested Number Positive Number Negative Relative Sensitivity (%) Relative Specificity (%) Combined Microscopy (Gold Standard) 1 290 169 146 100 100 Blood Film Microscopy (BF) 290 151 (14) a 139 (14) b 81.1 85.6 QBC 2 290 178 (40) a 112 (18) b 81.7 64.4 Antigen Detection ELISA 151 53 98 35.1 NA Western Blot Analysis 151 53 98 35.1 NA Micro-haematuria 290 47 (18) a 243 (107) b 17.2 93.2 Proteinuria 290 164 (62) a 126 (65) b 60.4 41.8 Bilirubinuria 290 17 (3) a 273 (140) b 8.3 91.1 1 Combined Test Results of Blood Film Microscopy and QBC. b Number of samples tested positive by Combined Microscopy. 2 Quantitative Buffy Coat Test. NA Not applicable because only microscopy positive A Number of samples tested negative by Combined Microscopy. samples were tested for antigens. University of Ghana http://ugspace.ug.edu.gh 248 Appendix 5b A publication on the relationship of parasitemia and anemia among patients with Plasmodium falciparum malaria in Ghana University of Ghana http://ugspace.ug.edu.gh 249 University of Ghana http://ugspace.ug.edu.gh 250 University of Ghana http://ugspace.ug.edu.gh 251 University of Ghana http://ugspace.ug.edu.gh 252 APPENDIX 6: ETHICAL CONSENT FORMS Appendix 6a ADULT CONSENT FORM FOR THE STUDY ENTITLED: RAPID URINE-BASED DIPSTICK TEST FOR DIAGNOSIS OF MALARIA IN INFANTS, PLACENTA AND LOW-RISK INDIVIDUALS FOR SUBJECTS AGED 16 YEARS AND OLDER UNIVERSITY OF GHANA KPONE ON SEA/GHANA ____________________________________________________________________________________________ ______ Information: to be read and translated to adults in their own mother tongue if necessary Adult’s Name: Age: Last name First name Middle initial Dear Sir/Madam, We invite you to take part in a research study sponsored by the School of Public Health, College of Health Sciences, University of Ghana and the Bill and Melinda Gates Institute of Population and Reproductive Health, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA. It is important that you understand several general principles that apply to everyone who takes part in this study: 1. Participation in the study is entirely voluntary. 2. Personal benefit to you may not result from taking part in the study, but knowledge gained may benefit others. 3. You may withdraw from the study at any time. Malaria is a sickness caused by very small disease causing organisms that can enter the body when mosquitoes bite you. They can cause fevers, headaches, body aches and weakness, and if not treated, can progress to severe illness, especially in children and pregnant women. When malaria is treated with proper medicines, it can be cured completely; but before a patient is given medicines to cure malaria a proper test has to be done to be sure that he/she has malaria. Presently, there are a number of methods that can be used to test for malaria but none of them uses urine even though it is easier to obtain and simpler to use. The main purpose of this study is to come out with a new method of detecting the presence of malaria using urine as a test material. The reason why we are inviting you to participate in this study is because we would like to learn more about the malaria disease that occurs in this community so that we can produce a new tool for detecting the disease early enough to cure it so as to prevent the disease from becoming severe and leading to death. The alternative to participating in this study is to not participate. If you agree to participate in this study, we will collect about seven dessertspoonful (50ml) of urine from you into a tube. In addition, we will stab, prick your finger with a Identifying Number __________________ Date __________________ Date __________________ Date __________________ University of Ghana http://ugspace.ug.edu.gh 253 lancet, special blade and collect few drops of blood into one small (capillary) tube and also, onto two glass microscope slides. Proteins from the urine will be used to produce the tool for detecting malaria whilst the blood will be examined for malaria causing organisms and also be measured to see if you have enough blood. The malaria test results will be ready within thirty minutes, and if you are found to have the small organisms that cause malaria and/ or do not have enough blood, we will refer you to the doctor for treatment. It is our policy to not discuss such information unless it has direct medical implications for treatment of malaria. We will store a part of the samples we collect in case there are additional tests we wish to perform in the future. These stored samples will be used only for studies related to malaria. Samples will be labelled with a code and will be accessed by study investigators only. You have the right to request that your samples should not be saved, and you may ask that these samples be destroyed at any time. The risks associated with this study are minimal. Drawing blood may cause discomfort and occasional bruising at the site; rarely, fainting or infection may occur. We will clean your finger with disinfectant before taking blood and will use a new lancet to prick your finger for the blood. If you wish to withdraw from the study, you may do so at any time. Although you will not receive any monetary payment for your participation, you will be given a small amount of non-monetary compensation for your time. The findings of this study may be reported at meetings or in medical journals, but your name will not be used in the reports, and the specific information we learn about you will not be shared with anybody except the study investigators. Do you have any questions about your participation in this study? If you have questions or concerns about your participation in this study at a later date, you may speak with one of our staff or you can ask the Chief of this area to send a message to Mr. Uri S. Markakpo at the School of Public Health, College of Health Sciences, University of Ghana, Legon. The doctors at the community clinic/ health centre can help you contact Mr. Markakpo. Yours Sincerely, Mr. Uri S. Markakpo Research Fellow Tel: 0246373898 If you agree to participate in this study, please put your signature or thumbprint below. Adult Signature or Thumbprint Investigator Signature Date University of Ghana http://ugspace.ug.edu.gh 254 Appendix 6b PARENTAL CONSENT FORM FOR THE STUDY ENTITLED: RAPID URINE-BASED DIPSTICK TEST FOR DIAGNOSIS OF MALARIA IN INFANTS, PLACENTA AND LOW-RISK INDIVIDUALS FOR SUBJECTS AGED 15 YEARS AND YOUNGER UNIVERSITY OF GHANA KPONE ON SEA/GHANA ____________________________________________________________________________________________ ______ Child’s Name: _________________________________________ Age: ___________________ Last name First name Middle initial Adult’s Name: _________________________________________ Relationship to child: ___________________ Last name First name Middle initial Information: to be read and translated to parents/ guardians in their own mother tongue if necessary. Dear Sir/Madam, We invite your child to take part in a research study sponsored by the School of Public Health, College of Health Sciences, University of Ghana and the Bill and Melinda Gates Institute of Population and Reproductive Health, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA. It is important that you understand several general principles that apply to everyone who takes part in this study: 1. Participation of your child in the study is entirely voluntary. 2. Personal benefit to your child may not result from taking part in the study, but knowledge gained may benefit others. 3. You may withdraw your child from the study at any time. Malaria is a sickness caused by very small disease causing organisms that can enter the body when mosquitoes bite you. They can cause fevers, headaches, body aches and weakness, and if not treated, can progress to severe illness, especially in children and pregnant women. When malaria is treated with proper medicines, it can be cured completely; but before a patient is given medicines to cure malaria, a proper test has to be done to be sure that he/she has malaria. Presently, there are a number of methods that can be used to test for malaria but none of them uses urine even though it is easier to obtain and simpler to use. The main purpose of this study is to come out with a new method of detecting the presence of malaria using urine as a test material. The reason why we are inviting your child to participate in this study is because we would like to learn more about the malaria disease that occurs in this community so that we can produce a new tool for detecting the disease early enough to cure it so as to prevent the disease from becoming severe and leading to death. The alternative to participating in this study is to not participate. Identifying Number__________________ Date __________________ Date __________________ University of Ghana http://ugspace.ug.edu.gh 255 If it is agreed that your child should participate in this study, we will collect about seven dessertspoonfuls (50ml) of urine from your child into a tube. In addition, we will prick your child’s finger with a lancet (special blade) and collect few drops of blood into one small tube and also, onto two glass microscope slides. Proteins from the urine will be used to produce the tool for detecting malaria whilst the blood will be examined for small organisms that cause malaria and also measured to see if your child has enough blood (anaemia). The malaria test results will be ready within thirty minutes, and if your child is found to have malaria causing organisms and/ or does not have enough blood, we will refer him/her to the doctor for treatment. It is our policy to not discuss such information unless it has direct medical implications for treatment of malaria. We will store a part of the blood and urine samples we collect in case there are additional tests we wish to perform in the future. These stored samples will be used only for studies related to malaria. Samples will be labelled with a code and will be accessed by study investigators only. You have the right to request that your child’s samples should not be saved, and you may ask that these samples be destroyed at any time. The risks associated with this study are minimal. Drawing blood may cause discomfort and occasional bruising at the site; rarely, fainting or infection may occur. We will clean your child’s finger with disinfectant before taking blood and will use a new lancet to draw the blood. If you wish to stop your child from participating in the study, you may do so at any time. Although you will not receive any monetary payment for your child’s participation, you will be given a small amount of non-monetary compensation for your time. The findings of this study may be reported at meetings or in medical journals, but your child’s name will not be used in the report, and the specific information we learn about your child will not be shared with anybody except the study investigators. Do you have any questions about your child’s participation in this study? If you have questions or concerns about your child’s participation in this study at a later date, you may speak with one of our staff or you can ask the Chief of this area to send a message to Mr. Uri S. Markakpo at the School of Public Health, College of Health Sciences, University of Ghana, Legon. The doctors at the community clinic/ health centre can help you contact Mr. Markakpo. Yours Sincerely, Mr. Uri S. Markakpo Research Fellow Tel: 0246373898 If you agree to have your child participate in this study, please put your signature or thumbprint below. Adult Signature or Thumbprint Investigator Signature Date University of Ghana http://ugspace.ug.edu.gh 256 Appendix 6c CHILD INFORMATION FORM FOR THE STUDY ENTITLED: RAPID URINE-BASED DIPSTICK TEST FOR DIAGNOSIS OF MALARIA IN INFANTS, PLACENTA AND LOW-RISK INDIVIDUALS Information: to be read and translated to children in their own mother tongue if necessary Explanation of the study to children in few words This study is to collect information about malaria in this community which has been selected as a model site for studies on diseases including malaria. The outcome of the studies will help to develop effective control methods to protect the people from these diseases, particularly, malaria. You will be asked to donate about seven dessertspoonful (50ml) of urine. Also, a fresh sterile lancet (special blade) will be used to prick your finger to take a few drops of blood. The urine sample will be used to produce a new tool for determining whether you have malaria or not. If this new tool is produced urine may be used to check for malaria instead of blood. Also, qualified health personnel in the laboratory will examine the blood collected for small organisms that cause malaria and to see if you have enough blood. The malaria test results will be ready within thirty minutes, and if you are found to have the organisms that cause malaria and/ or do not have enough blood, we will refer you to the doctor for treatment. Apart from a minor pain at the point where your finger will be stabbed with the lancet, urine and blood sample collection, will not have any bad effect on you. You will be assigned a code that will be used to identify you and all information gathered from you will be considered confidential. Informed consent has been obtained from your parent/guardian, but you can decide not to take part in the study if you do not want to. Also, if at any time after the study has begun you decide not to participate or allow your samples to be used in the study anymore, you are free to withdraw or inform us immediately without any further discussion. Withdrawal from the study or refusal to participate will not have any adverse consequence on you or deny you with any rights or benefits you are already enjoying in this community or elsewhere. If you agree to participate in this study, please allow your name to be written in the space provided below. Name of Child: Last name First name Middle initial Translated by: Translation witnessed by: Signature: Name: Designation: University of Ghana http://ugspace.ug.edu.gh 257 APPENDIX 7: ETHICAL CLEARANCE CERTIFICATE University of Ghana http://ugspace.ug.edu.gh 258 APPENDIX 8: TECHNICAL CHALLENGES ENCOUNTERED IN THE COURSE OF THE STUDY AND HOW THEY WERE CIRCUMVENTED Following the obtainment of funding, other problems encountered include:  Lack of space for placement of freezers and refrigerators for storage of samples at NMIMR. To overcome this problem, some broken down freezers occupying space in the laboratory were refurbished and used.  Removal of a voltage stabilizer from a storage freezer for my research by an unknown person which resulted in the breakdown of the freezer with associate loss of some reagents and processed samples which destabilized work for some time. The stolen stabilizer has not been replaced. An alternative freezer belonging to Prof. I. A. Quakyi, containing left over reagents was evacuated and is now being used for storage of reagents.  Loss of a HAT selection reagent, a critical reagent for production of monoclonal antibodies (which are essential for development of the urine-based dipstick test) from my storage freezer at the Noguchi Memorial Institute for Medical Research (NMIMR) which took me about three (3) months to get a replacement. To circumvent this challenge, a substitute for this reagent was procured for the work.  Breakdown of the two (2) carbon dioxide (CO2) incubators for culture of hybridoma cells needed for production of monoclonal antibodies. This technical challenge caused cell fusion to be suspended for more than 3 months. An alternative incubator belonging to Prof. Quakyi was later moved to Noguchi for continuation of work until the problem with the first incubator had been resolved.  Closure of my working laboratories at the NMIMR facility for refurbishment for over 3 months.  Shut down of the biological safety cabinet for cell culture for maintenance which took 3 weeks to complete.  Breakdown of CO2 cylinders for tissue culture which took over 3 months to repair and refilled by AirLiquide, Tema, before continuation of work.  Failure of cell fusion to yield hybridoma cells which took more than 2 months to overcome.  Loss of hybridoma cells in culture to bacterial contamination which took 2 months to recover.  Delay in the release of project funds. This challenge has now minimized University of Ghana http://ugspace.ug.edu.gh