University of Ghana http://ugspace.ug.edu.gh September 1, 2022 The Dean School of Graduate Studies Univertsity of Ghana Legon Dear Sir, CORRECTED THESIS FOR FINAL SUBMISSION – BENJAMIN AGRAH (10637893) I have gone through the final thesis of the above named MPhil student. I can certify that he has effected all the corrrection in his thesis as instructed by his examiners for final submission. Thank you. Yours faithfully, Dr. Bartholomew Dzudzor Head of Department University of Ghana http://ugspace.ug.edu.gh COMMENTS STUDENT’S RESPONSE TO COMMENTS The background is good except that the The background information has been re-written and introduction is written without a paragraph. sectioned into six (6) paragraphs. Also, the entire thesis has been sectioned into sub-headings. The problem statement under investigation is All grammatical errors in the problem statement have clear albeit few grammatical errors. been corrected. Most factual statements were without citations, All factual statements have been cited accordingly and also summarize malaria diagnostic sub-topics in the malaria diagnostic sub-topics have been the literature review. summarized into one sub-heading The candidate worked on archived samples of Sub-section 3.7.1 has been re-written to acknowledge which parasitaemia were already established. This the fact that microscopy was previously done before section must be re-written. the commencement of this study. Categorize results according to specific objectives All results have been sectioned logically according to the specific objectives of the study and all figures have been cross-checked. The description of demographics should come The description of demographics of the study has been before the demographics table. changed to precede the table of demographics. The discussion should have sub-headings. The discussion section has been categorized into four sub-headings Provide more information on Capillary Detailed information on capillary electrophoresis and electrophoresis and add why the use of ELISA how it is used to separate the various HB fractions was warranted in the study. (Sub-headings 2.13). Also, a detailed information on ELISA and why it was used in this study has been added. Some cited references are not in the final All citations have been included in the final reference reference section. The candidate is advised to go and all incomplete references have been fixed. through the reference section and fix incomplete references as well. Student should clarify which health facilities were The names of the health facilities have been included chosen for the study. and can be found in sub-heading 3.1. Also clarify if two sets of age-specific population The age range for this study was 2-89 years and there were used. was no age matched control group. Why didn’t the candidate read any microscopy The microscopy slides were already read by two slide? WHO-certified malaria microscopists and any disagreement in reading was re- examined by a third microscopist. Moreover, the microscopy slides were not readily accessible. Signed: Nii Ayite Aryee (Principal Supervisor) Linda Eva Amoah (Co-Supervisor) University of Ghana http://ugspace.ug.edu.gh ANTIBODY RESPONSES GENERATED AGAINST P. FALCIPARUM AND A. GAMBIAE ANTIGENS IN SUSPECTED MALARIA PATIENTS WITH VARIANT HAEMOGLOBIN GENOTYPES BY BENJAMIN AGRAH (10637893) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON, IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL IN MEDICAL BIOCHEMISTRY DEGREE JULY, 2021 i University of Ghana http://ugspace.ug.edu.gh DECLARATION I performed the experimental work described in this thesis at the Department of Immunology of the Noguchi Memorial Institute for Medical Research and the SickleGenAfrica Lab at the University of Ghana Medical School, Korle Bu, under the supervision of Dr. Nii Ayite Aryee and Dr. Linda Eva Amoah. Candidate BENJAMIN AGRAH (10637893) Supervisor DR. LINDA EVA AMOAH (Department of Immunology, NMIMR) Principal Supervisor DR. NII AYITE ARYEE (Department of Medical Biochemistry, UGMS) ii University of Ghana http://ugspace.ug.edu.gh DEDICATION I dedicate this work to my family, especially my hardworking mum, for her immense contribution to my education and her ceaseless prayers for me. iii University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGMENT My deepest gratitude goes to God Almighty for sustaining me through the challenging moments I faced. Special thanks go to my supervisors, Dr. Linda Amoah of the Immunology Department, Noguchi Memorial Institute for Medical Research (NMIMR) for her unfailing scholarly advice and the substantial financial support she provided for this study and Dr. Nii Ayite Aryee of the Department of Medical Biochemistry, College of Health Sciences, University of Ghana, for his time, guidance, and support My heartfelt thanks go to Dr. Festus Acquah of the Department of Immunology (NMIMR) for leading me through the optimization of my ELISA and the analysis of my research. A word of gratitude also goes to Professor Ben Gyan, Head of the Department of Immunology at NMIMR, for enabling me to work in the department. I would also like to thank the rest of the staff of the Immunology Department for their contribution. I am also very grateful to all the staff of the SickleGenAfrica Lab for allowing me to use their laboratory and providing the necessary assistance. To Dr. William Kudzi, Kelvin, and Joseph, I say a big thank you for your contributions in the course of my work. iv University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS DECLARATION .............................................................................................................. ii DEDICATION .................................................................................................................iii ACKNOWLEDGMENT .................................................................................................. iv TABLE OF CONTENTS .................................................................................................. v LIST OF TABLES .......................................................................................................... viii LIST OF FIGURES ....................................................................................................... ..ix LIST OF ABBREVIATIONS .......................................................................................... x ABSTRACT .................................................................................................................... xii CHAPTER 1 ..................................................................................................................... 1 INTRODUCTION 1.1 Background ............................................................................................................ 1 1.1.1 Problem Statement ............................................................................................ 3 1.1.2 Justification ....................................................................................................... 3 1.1.3 Research Hypothesis ......................................................................................... 4 1.1.4 Aim of Study..................................................................................................... 4 1.1.5 Specific Objectives ............................................................................................ 4 CHAPTER 2 ..................................................................................................................... 5 LITERATURE REVIEW .............................................................................................. 5 2.1 The Burden of Malaria ......................................................................................... 5 2.2 The life cycle of Plasmodium falciparum ............................................................. 6 2.3 Case definition of clinical malaria ........................................................................ 7 2.3.Signs and symptoms……………………………………………………………….8 2.4 Haemoglobin Variants……………………………………………………………..9 2.4.1 Malaria and Haemoglobin variants .................................................................. 11 2.4.2 Erythrocytes and Plasmodium falciparum........................................................ 12 2.5 Malaria immunity ............................................................................................... 13 2.5.1 Innate immune response………………………………………………………...…13 2.6 Acquired immunity………………………………………………….………………14 2.6.1 Humoral Immune response…………………………………………………….….14 2.6.2 Mechanism of antibody repose…………………………………….………………14 2.6.3 Cell-mediated Immunity…………………………………………………………...15 v University of Ghana http://ugspace.ug.edu.gh 2.6.4 Mechanism of cellular immune response…………………………………….……16 2.7 Haemoglobin variants restrict P. falciparum growth in RBCs ............................. 17 2.8 Haemoglobin variants interference with mechanisms of P. falciparum Malaria .................................................................................................................... 17 2.9 Haemoglobin variants influence the Innate host defense responses to P. falciparum .......................................................................................................... 18 2.10 Haemoglobin variants enhance the adaptive immune responses to P. falciparum ………………………………………………………………………………...20 2.11Malaria Diagnostics............................................................................................ 21 2.12 Malaria serology ............................................................................................... 22 2.12.1 Asexual Stage Antigen - PfEBA-175………………………………………….23 2.12.2 Sexual Stage Antigen - Pfs230……………………………………. ………….24 2.12.3 Salivary Gland antigen- gSG6-P1………………………………………….…26 2.13 Capillary electrophoresis .................................................................................. 26 2.14 Enzyme-Linked Immunosorbent Assay (ELISA)……………………………....30 CHAPTER 3 ................................................................................................................... 31 MATERIALS AND METHODS ................................................................................. 31 3.1 Study type and description of study sites................................................................ 31 3.1.1 Subjects/study population ................................................................................ 31 3.2 Inclusion Criteria ................................................................................................... 31 3.3 Exclusion Criteria .................................................................................................. 31 3.4 Sample size determination ..................................................................................... 32 3.5 Ethical Consideration ............................................................................................. 33 3.6 MATERIALS ........................................................................................................ 33 3.6.1 Reagents ......................................................................................................... 33 3.7 METHODS ........................................................................................................... 33 3.7.1 Examination of malaria Parasite by Microscopy .............................................. 33 3.7.2 Indirect ELISA ................................................................................................ 34 3.7.3 Haemoglobin Phenotype determination” .......................................................... 35 3.8 Data analysis ......................................................................................................... 35 CHAPTER 4 ................................................................................................................... 36 RESULTS ........................................................................................................ 36 vi University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE ............................................................................................................ 51 DISCUSSION, CONCLUSION, AND RECOMMENDATION .................................. 51 5.1 DISCUSSION ....................................................................................................... 51 5.2 CONCLUSION ..................................................................................................... 57 5.3 RECOMMENDATION ......................................................................................... 58 REFERENCES ............................................................................................................... 59 APPENDICES ................................................................................................................ 84 vii University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 1: Demographic Characteristics of the Study Participants Grouped Regionally. .... 36 Table 2: Comparison of the IgG Levels for gSG6-P1 in Malaria Positive and Negative Individuals at Each Region Using Mann Whitney U Test ................................................ 37 Table 3: Comparison of the IgG Levels for Pfs-230 in Malaria Positive and Negative Individuals at Each Region Using Mann Whitney U Test ................................................ 39 Table 4: Comparison of the IgG Levels for EBA 175 3R in Malaria Positive and Negative Individuals at Each Region Using Mann Whitney U Test ................................................ 41 Table 5: National Comparison of the Levels for IgG gSG6-P1, Pfs230, and EBA 175 3R in Malaria Microscopy Positive and Negative Individuals Using the Mann Whitney U Test. . ...................................................................................................................................... 42 Table 6: Kruskal Wallis Variational Test Between the Haemoglobin Variants and the Concentration of IgG’s measured in Microscopy Positive Samples ................................. 44 Table 7: Seroprevalence of IgG Antibodies for gSG6-P1, Pfs230, and EBA 175 3R antigens in Microscopy Positive and Negative Samples Stratified Across Gender, Age, and HB Phenotype ....................................................................................................................... 49 viii University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure 1: Life Cycle of Plasmodium falciparum (Bousema et al.,2014) ............................ 7 Figure 2: Capillary electrophoresis system; CAPILLARYS 2 FLEX PIERCING ........... 29 Figure 3: A typical separation profile obtained with the Capillarys 2 Flex Piercing analyzer. ......................................................................................................................... 29 Figure 4: Occurrence of Haemoglobin Variants Observed Among Malaria Positive Individuals in Eight Regions ........................................................................................... 43 Figure 5: Seroprevalence of IgG Antibodies for gSG6-P1, Pfs 230, and EBA 175 3R antigens in Microscopy Positive and Negative Samples .................................................. 45 ix University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS. Ab Antibody CDC Centre For Disease Control and Prevention DNA Deoxyribonucleic Acid EBA Erythrocyte-binding Antigen ELISA Enzyme-Linked Immunosorbent Assay GHS Ghana Health Service GLURP Glutamate Rich Protein gSG6-P1 gambiae Salivary Gland Hb Haemoglobin HO Heme Oxygenase HRPO Horseradish Peroxidase IFN Interferon Ig Immunoglobulin IgG Immunoglobulin G IgM Immunoglobulin M IgE Immunoglobulin E IL Interleukin iRBCs Infected Red Blood Cells NMCP National Malaria Control Program OD Optical Density PBMC Peripheral Blood Mononuclear Cell Pf Plasmodium falciparum PfEMP Plasmodium falciparum Erythrocyte Membrane Protein x University of Ghana http://ugspace.ug.edu.gh PfHRP Plasmodium falciparum Histidine Rich Protein pLDH Parasite Lactate Dehydrogenase RBC Red Blood Cell RDT Rapid Diagnostic Test SPSS Statistical Package for Social Science TH T Helper Cell TH1 T Helper 1 TH2 T Helper 2 TGF Tumor Growth Factor TNF Tumor Necrosis Factor WHO World Health Organization xi University of Ghana http://ugspace.ug.edu.gh ABSTRACT HbAS and HbAC are known to be protective against P. falciparum infection, but it is unclear how this protection is conferred in malaria symptomatic patients in Ghana. Theoretically, HbAS and HbAC protect against P. falciparum malaria by improving naturally acquired immunity to the parasite. Also, Immunoglobulin G (IgG) has played a significant role in blood parasite clearance in individuals infected with P. falciparum, suggesting the protective mechanism of the acquired immunity. This study investigated haemoglobin genotypes and their effects on IgG levels in symptomatic malaria. This research was a nested archival cross-sectional study that enrolled 600 symptomatic malaria patients aged between 2 to 89 from the ten regions in Ghana. Parasite species and density were determined, followed by haemoglobin phenotyping for malaria microscopy-positive patients. An indirect enzyme-linked immunosorbent assay (ELISA) was performed on all the samples to examine the differential effects of exposure to Anopheles mosquitoes (gSG6-P1), sexual stage malaria parasites (Pfs230), and asexual stage malaria parasites (EBA 175 3R). The haemoglobin variants observed among malaria microscopy-positive patients in eight regions of Ghana were HbAA, HbAC, HbSC, HbAS, HbSS, HbCC, and HbAF. In conclusion, there were significant differences in the total concentration of anti-EBA 175 3R and anti-gSG6-P1 antibodies in malaria negative and positive microscopy samples. Although a significant association was established between the concentrations of IgGs measured against the various antigens in different haemoglobin variants in malaria microscopy positive samples, it was clear that the number of participants with IgG against gSG6-P1 antigen was significantly greater in HbAA participants than in the other haemoglobin variants. Yet the same assessment could not be made for the sexual stage antigen (Pfs230) and the erythrocyte binding antigen (EBA 175 3R). In general, no xii University of Ghana http://ugspace.ug.edu.gh significant relationships were established between the influence of age, gender, and haemoglobin variants on the anti-Pfs230, anti-EBA 175 3R, and anti-gSG6- P1 antibodies. xiii University of Ghana http://ugspace.ug.edu.gh CHAPTER 1 INTRODUCTION 1.1 Background Malaria poses a significant health challenge in many countries worldwide, particularly in the WHO African region. In 2018, an estimated 228 million malaria cases occurred worldwide, while 213 million were recorded in the World Health Organization (WHO) African Region (WHO, 2019). Malaria is an infectious disease caused by a Plasmodium genus unicellular haemosporozoan parasite, four of which cause disease in humans, namely P. vivax, P. malariae, P. ovale, and P. falciparum. It has recently been shown that Plasmodium knowlesi and P. cynomolgi, identified as simian malaria parasites, also cause life-threatening human malaria (Cox-Singh, 2008; Luchavez, 2008; Ta, 2014). These parasites are transmitted to humans through the infective bite of a female Anopheline mosquito, which serves as the vector. Nevertheless, P. falciparum infection is primarily associated with the most severe forms of the disease, such as cerebral malaria and severe malaria anemia (Miller,1994). P. falciparum is the most prevalent malaria parasite in the WHO African Region, accounting for 99.7 % of estimated malaria cases recorded in 2018 (WHO, 2019). The global estimated malaria deaths documented by the WHO were 405,000 in 2018 (WHO, 2019). While high-risk groups include persons with less evolved immune systems, children under five years of age rarely exposed to infection in endemic areas are also at risk (Miller, 1994). P. falciparum infections can cause a wide range of diseases. There is some non-sterile clinical immunity after repeated parasite exposure (Bull, 1998). 1 University of Ghana http://ugspace.ug.edu.gh Recent research shows that development has slowed after an unparalleled period of success in global malaria prevention. In 2020, between January and March, Ghana recorded approximately 1,001,070 malaria cases. And out of this number, 21,201 were children under five years old, and 28,764 were pregnant women (Shretta, 2020). Several efforts have been taken to eradicate it, but it continues to spread over the globe, even in previously exterminated places. The most challenging aspect of malaria control is the emergence of drug-resistant variants of the parasite, particularly P. falciparum (Akanbi, 2014). In endemic areas, characteristics offering partial resistance to infection or illness progression are selected, and this has had and continues to have a widespread impact on human population genetics (Amoah, 2018). It is commonly known that haemoglobin (Hb) variants, particularly the sickle cell trait (HbAS), have been validated for several genetic polymorphisms that confer relative resistance to malaria (Aidoo, 2002). Likewise, alpha-thalassaemia protects from severe malaria in Papua New Guinea (Allen, 1997). Among the mechanisms involved are resistance to parasite invasion, reduced intracellular proliferation, altered neoantigen production, and increased vulnerability to oxidative stress (Wilkinson, 1997). In Gabon, HbAS was linked to a significant proportion of polyclonal P. falciparum infections, indicating a high multiplicity of infection (Lassana Konaté, 1999) but not in Senegal. The protective effect of HbAS has been connected to some modification of the immune response against malaria, but new findings demonstrate that it involves rapidly acquired immunity to the parasite; nonetheless, the mechanisms of protection remain unknown. 2 University of Ghana http://ugspace.ug.edu.gh 1.1.1 Problem Statement Malaria is a major public health concern, with a high incidence of mortality in Ghana (Shretta, 2020). There is a fascinating co-evolutionary process between the Plasmodium species and humans. Malaria-affected populations have a variety of haemoglobin variations that may work together to diminish Plasmodium virulence in the human body, resulting from distinct geographical originating effects. In the search for immunological surrogates of immunity against malaria, a plethora of research has focused on antibody levels without determining the phenotype of haemoglobin associated, which is undoubtedly a critical parameter in measuring and monitoring immunity to malaria. There is a need to know whether the haemoglobin variants affect antibody production in malaria microscopy-positive individuals across Ghana. Although the stories of HbS and HbC have provided fascinating insights into evolutionary biology, a thorough knowledge of their malaria-protective mechanisms is required before these lessons can be applied. This situation can be considered among the reasons why malaria vaccine development has been a challenging task for scientists 1.1.2. Justification In this study, it will be essential to elucidate the influence of haemoglobin variants on immune response, thus, adding more insight into malaria surveillance in Ghana and the development of a more potent drug regime or vaccine to curb this menace. Haemoglobin variants are relatively common in Ghana, many of which have been described (Danquah, 2010). Several antibodies identified as key to malaria protection and alleviating symptoms of the disease lack rigorous information on the influence of haemoglobin variants on IgG levels in malaria symptomatic patients across Ghana. This research investigates the relationship between antibodies against 3 University of Ghana http://ugspace.ug.edu.gh malaria antigens, EBA-175 (Region III), Pfs 230, and gSG6 –P1 and haemoglobin genotype in malaria-infected persons. This information will equip researchers and health workers to design and administer vaccines and drugs based on an individual’s haemoglobin phenotype leading to an individual-centered treatment, thus pharmacogenomics in malaria treatments in Ghana. 1.1.2 Research Hypothesis The levels of IgG elicited against malaria parasites and vector antigens in suspected malaria patients with varying haemoglobin genotypes differ. 1.1.3 Aim of Study The study investigated IgG responses to three malaria antigens, EBA 175 3R, Pfs230, and gSG6-P1, and the effects of haemoglobin variants on IgG levels in malaria microscopy- positive symptomatic patients across Ghana. 1.1.4 Specific Objectives 1. To determine and compare IgG responses to two malaria parasite antigens (EBA 175 3R, Pfs230) and one mosquito vector antigen, gSG6-P1, in plasma samples collected from suspected malaria patients across Ghana. 2. To determine the different haemoglobin genotypes in microscopy-positive samples across Ghana 3. To determine the seroprevalence of two malaria parasite antigens (EBA 175 3R, Pfs230, and one mosquito vector antigen (gSG6-P1) in microscopy positive and negative samples collected across Ghana. 4. To elucidate the relationship between haemoglobin variants and malaria anti-IgG levels in microscopy-positive samples across Ghana 4 University of Ghana http://ugspace.ug.edu.gh CHAPTER 2 LITERATURE REVIEW 2.1 The Burden of Malaria Malaria remains an overwhelming problem in developing countries, affecting the economy and productivity in endemic areas. Globally, 228 million malaria cases were reported in 2018, compared to 251 million cases in 2010 and 231 million in 2017 (WHO, 2019). Also, 93 % of these cases occurred in the WHO African region, followed by 3.4 % for the WHO South-East Asia region and 2.1 % for the WHO Eastern Mediterranean region (WHO, 2019). About 19 countries in sub-Saharan Africa and India accounted for nearly 85 % of the global burden of malaria (WHO, 2019). Between 2010 and 2018, the global malaria incidence rate dropped from 71 to 57 cases per 1000 people at risk (WHO, 2019). However, the rate of change slowed dramatically from 2014 to 2018, dropping to 57 in 2014 and remaining at similar levels through 2018 (WHO, 2019). P. falciparum is the most common malaria parasite in the WHO African Region, accounting for 99.7% of malaria cases estimated in 2018 (WHO, 2019). Globally, malaria deaths in 2018 were estimated to be 405,000 compared with 2017 and 2010, which were 416,000 and 585,000, respectively (WHO, 2019). Further, children under five years old, the most vulnerable group, accounted for 67 % of malaria deaths worldwide (WHO, 2019). Africa saw the most significant absolute decrease in deaths from malaria, from 533,000 to 380,000 from 2010- 2018 (WHO, 2019). However, despite progress, the mortality rate has decreased since 2016; though the disease burden of 155 million in high-impact countries saw significant decreases from 2010 to 2018, a higher number of cases occurred in 2018 compared to previous years (WHO, 2019). In Africa, Ghana and Nigeria reported the highest absolute increases in malaria cases in 2018 compared with 2017 (WHO, 2019). 5 University of Ghana http://ugspace.ug.edu.gh The 2018 malaria burden was similar to that of 2017 in all other countries, except Uganda and India, where reductions of 1.5 and 2.6 million have been reported (WHO, 2019). But in Ghana, there was a notable increase (8 %) in malaria cases in Ghana (WHO, 2019). Finally, the economic burden of malaria in sub-Saharan Africa is unmistakable; much of the world's poorest countries are in sub-Saharan Africa, where malaria is endemic (Sachs, 2002). The worldwide distribution of per-capita gross domestic product reveals a robust association between malaria and poverty (Asante, 2003). In contrast, malaria-endemic countries often have lower levels of economic growth than non-endemic countries (Asante, 2003). The effect of malaria on real GDP growth in Ghana is negative and decreases by 0.41 % for every 1 % increase in malaria morbidity (Asante, 2003). 2.2 The life cycle of Plasmodium falciparum During a blood meal, infected female Anopheles spp. mosquitoes inject sporozoites into the bloodstream, causing infections in humans. This is accompanied by the release of merozoites into the bloodstream, which initiates the multiplication stage of asexual parasites (see figure 1). The length of an infection in the blood system is highly variable: several infections are cleared early, while others remain for several months (Felger, 2012). A fraction of merozoites form sexual gametocytes, the only parasite forms capable of transmitting from humans to mosquitoes. In the bone marrow, immature gametocytes (those in stages I – IV) are sequestered, and only mature gametocytes (stage V) circulate in the peripheral blood. In peripheral blood, the number of mature gametocytes is usually less than 100 gametocytes per μl of blood (Drakeley, 2006), and the vast majority are present at submicroscopic densities. After mosquito ingestion, each gametocyte forms one female macrogamete or a maximum of eight male microgametes. Gamete fusion in the mosquito midgut produces a zygote that grows into a motile ookinete that can infiltrate the midgut wall and form oocysts. 6 University of Ghana http://ugspace.ug.edu.gh The oocysts grow over time and burst to release sporozoites that migrate to the salivary gland of the mosquito, from which they may infect humans during the next meal (Baidjoe, 2013). gSG6-P1 EBA 175 Pfs 230 Figure 1: Life Cycle of Plasmodium falciparum (Bousema et al.,2014) 2. 3 Case definition of clinical malaria Clinical disease caused by the parasite is due to the asexual replication of parasites in the blood stage, so much attention has been paid to the erythrocytic stage to identify antigens intended for defensive immune responses. Clinical symptoms of P. falciparum infection include chills, fatigue, headache, nausea, vomiting, and diarrhea. The primary complications of P. falciparum malaria in children include cerebral malaria, extreme anemia, seizures, and respiratory distress. Clinical malaria description is generally based on microscopic identification of malaria. 7 University of Ghana http://ugspace.ug.edu.gh Parasitemia, fever, and febrile temperature > 37.5°C. Malaria fever is caused by the release of parasite toxins when schizont-infected red blood cells rupture to release merozoites (Kwiatkowski, 1989). Thus, malaria is said to be directly caused by the rapturing of the red blood cells that contribute to febrile temperatures. Using febrile fever > 37.5 °C and parasitemia at various rates results in a highly susceptible and precise case description of malaria (Armstrong, 1994). However, individuals in highly endemic areas may have parasites without fever or other symptoms associated with malaria. 2. 3 Signs and symptoms Asexual erythrocytic or blood-stage parasites are responsible for all clinical symptoms associated with malaria (CDC, 2019). The time between the contagious bite and the appearance of clinical signs is around 9 to 14 days (Garrison, 2015). Malaria typically causes recurrent attacks or paroxysms, each of which has three stages: chills, fever, and sweating. Along with chills, a person is likely to have headaches, malaise, exhaustion, muscle aches, and sometimes nausea, vomiting, and diarrhea. The body's temperature increases within an hour, and the skin feels hot and dry (Bartoloni, 2012). And, as the body's temperature falls, a drenching sweat begins. The person, feeling tired and weak, is likely to fall asleep (Bartoloni, 2012). Typical paroxysm, however, is present in only a minority of cases, especially in P. falciparum malaria (Hall, 1977). Clinical attacks may be triggered by overwork, emotional shock, pregnancy, delivery, illness, surgical operation, or general anesthesia. The onset can be sudden and severe, particularly in children and unprotected non-immune individuals, with death following in a matter of time (Felger, 2012). Malaria symptoms can be classified into two categories: uncomplicated and severe malaria. Uncomplicated malaria is diagnosed when symptoms are present, but no clinical or laboratory signs indicate a severe infection or dysfunction of vital organs. Individuals with uncomplicated malaria can develop severe malaria if the illness is not treated or their immunity 8 University of Ghana http://ugspace.ug.edu.gh to the disease is insufficient or non-existent. Uncomplicated malaria symptoms usually last 6-10 hours and come in cycles every other day, while some parasite strains might cause a longer cycle or mixed symptoms. Symptoms are often flu-like and may be undiagnosed or misdiagnosed in areas where malaria is less common. Symptoms of uncomplicated malaria include cold, hot, and sweating: the sensation of cold, shivering, fever, headaches, and vomiting (seizures sometimes occur in young children), sweats followed by a return to normal temperature, with tiredness (CDC, 2019). Severe malaria is defined by clinical or laboratory evidence of vital organ dysfunction. This form can be fatal if left unchecked. Symptoms of severe malaria include fever and chills, impaired consciousness, and prostration (CDC, 2019). 2. 4 Haemoglobin Variants Haemoglobin is an erythrocyte protein that comprises four polypeptide globin chains looped around a haem molecule. It is responsible for bringing oxygen from the lungs and carbon dioxide from the tissues to the lungs. The globin chains are encoded on chromosome 11 and chromosome 16 by their respective genes and are known to have multiple alleles (Weatherall, 2011). Many of these alleles suffer point mutations in the DNA sequence, which leads to single amino acid substitution in the moiety of globin, resulting in the production of haemoglobin variants. The abnormal haemoglobin genotype occurs when an affected person inherits mutated globin genes from both parents. Abnormal haemoglobin genotypes are inherited in an autosomal codominant pattern and occur through different combinations. Several hundred unusual haemoglobin genotypes have been found; however, only a few are typical and cause significant public health problems in many parts of the world (Weatherall, 2011). More than 1,000 human haemoglobin variants have been discovered with single amino acid substitutions that contribute to physiological consequences of varying severity (Giardine, 2013). 9 University of Ghana http://ugspace.ug.edu.gh HbS is the most prevalent pathological haemoglobin mutation leading to global substitution (Weatherall, 2011). HbS is Africa's most prevalent pathological form of haemoglobin, while HbD and HbE are among Indian and Southeast Asian populations (Walker, 2015). HbA2 fractions and fetal haemoglobin (HbF) may be increased in thalassemia, a disorder that affects the synthesis of the haemoglobin alpha or beta-globin chains. Beta-thalassemia can also occur when HbS and HbE are present. A combination sickle/beta-thalassemia phenotype occurs most commonly in the geographical region of the Mediterranean Sea (Walker, 2015). The sickle haemoglobin mutation is a structural variant of normal adult haemoglobin A that results from a single-point mutation in which the amino acid, glutamic acid is substituted by valine at position 6 of the haemoglobin A β- globin chain (Orkin, 2019). Carriers or heterozygous (AS) individuals inherit the HbS allele from one parent and HbA allele from the other but are typically asymptomatic, while homozygous (SS) individuals inheriting HbS alleles from both parents have the genotype of the disease and suffer from sickle cell anemia, which often leads to acute and chronic complications (Williams., 2011). Due to its haemolytic influence, the homozygous (SS) sickle haemoglobin type is called sickle cell anemia (SCA), which is generally the most frequently inherited genetic condition in the world and the most common form of sickle cell disease. The most severe types of sickle cell disease (SCD) include people with HbSS and HbSβ ° (Creary, 2007; Rees, 2010). Haemoglobin variants are likely to be relatively common in Africa, and several hundred have been described (Schaefer, 2016). 10 University of Ghana http://ugspace.ug.edu.gh Haemoglobin mutations can cause a wide range of phenotypic outcomes, including protein instability, cell lysis anemia, and microvascular circulation occlusion leading to ischemia, infarction, and chronic haemolytic anemia (Kanter, 2013; Bissé, 2017). In a study carried out on haemoglobin variants among patients attending Ho Teaching Hospital in Ghana, the prevalence of HbAS, HbA, HbSC, HbS, HbAC, HbSF, HbC, and HbAF among the study population for three years was estimated at 40.3%, 37.3%, 7.9%, 6.2%, 5.2%, 2.6%, and 0.3%, respectively (Awaitey, 2020). 2. 4. 1 Malaria and Haemoglobin variants While existing treatments have lessened morbidity in some cases, we need to increase our fundamental understanding of P. falciparum malaria pathogenesis to discover molecular and cellular targets for next-generation therapeutic and preventive strategies. Due to the complex tangle of parasite virulence factors, host susceptibility features, and innate and adaptive immune responses that affect the development of various malaria syndromes, mechanisms of falciparum malaria pathogenesis remain unknown (Bejon, 2008; O'Meara, 2008; Amaratunga., 2011). The risk of severe falciparum malaria in Sub-Saharan African children is reduced by 90% and 70% by heterozygous haemoglobin (HbAS) and homozygous haemoglobin C, respectively (Taylor, 2012). These structural haemoglobin variants do not protect against P. falciparum infection, suggesting that they interfere with the specific molecular mechanisms responsible for the morbidity of falciparum malaria (Taylor, 2012). By isolating these pathogenic processes and resolving the Gordian knot of malaria pathogenesis, haemoglobinopathies offer an attractive "natural experiment" to identify molecular correlations of clinical morbidity. These correlations may be capable of being exploited by future parasiticidal, adjunctive, or preventive therapies to yield targets for a new "Alexandrian solution" to the global P. falciparum malaria problem. 11 University of Ghana http://ugspace.ug.edu.gh 2. 4. 2 Erythrocytes and Plasmodium falciparum Erythrocytes are critical to the spread of malaria parasites. After inoculation in humans by a mosquito and a brief, clinically silent incubation in the liver, P. falciparum parasites enter the erythrocytic stage of their life cycle (Taylor, 2013). Parasites successively enter and break away from their host RBCs throughout this time, causing malaria signs and symptoms; while developing within RBC, the parasite transfers proteins to the RBC surface that mediate binding to extracellular host receptors, and it enables the parasite to sequester in the placenta, brain, and virtually any other organ. RBC variants are produced from some of the most common human genetic polymorphisms; their widespread prevalence has been assumed to result from their evolutionary selection by severe, life-threatening falciparum malaria (Taylor, 2013). Clinical data for several common haemoglobin disorders strongly supports this natural selection, and major haemoglobinopathies result from molecular lesions that either reduce the production of α or β globins or encode single amino acid substitutions in β-globin (in HbS, HbC, and haemoglobin E) (Weatherall., 2000). The most severe haemoglobinopathies, HbSS homozygosity, and thalassemia major are typically incompatible with early childhood life without sophisticated medical care. Other haemoglobin traits such as HbAS, HbAC, HbCC, HbAE, HbEE, and minor thalassemias are associated with essentially average life span and far less directly attributable morbidity. Remarkably, these simple polymorphisms provide dramatic levels of protection against complex diseases: for HbAS, replacing glycine with the amino acid at position 6 in only one of two β-globin chains reduces the risk of severe falciparum malaria in children by approximately 90 % (Taylor, 2013). 12 University of Ghana http://ugspace.ug.edu.gh The current comprehension of P. falciparum malaria pathogenesis suggests four general hypotheses for investigating the nature of malaria protection by haemoglobinopathies 1. Restriction of RBC invasion or intraerythrocytic parasite growth. 2. Interference with parasite-derived mediators of pathogenesis. 3. Modulation of innate host responses. 4. Enhancement of the host's adaptive immune clearance of parasite-infected RBCs (iRBCs). 2. 5 Malaria immunity 2. 5. 1 Innate immune response The innate immune system provides non-specific protection through monocytes, macrophages, dendritic cells, natural killer cells, eosinophils, neutrophils, mast cells, complement, and acute phase proteins. This also requires physical defenses such as epithelial layers and antimicrobials on these surfaces (Taylor, 2013). Neutrophils, mononuclear phagocytes, and natural killer cells play a role in malaria infections in innate immunity. Natural killer cells are involved in erythrocytes infected with P. falciparum lyses in vitro. They contain cytokines such as interferon (IFN-y) that cause macrophages to phagocytose invasive foreign particles (Orago, 1991; Artavanis-Tsakonas, 2002). In addition, innate immune mechanisms by natural killer cells lead to the stimulation of IFN, which limits the initial phase of parasite replication; this has also been demonstrated in studies done on murine malaria (Good, 1999; Fell, 1998). 13 University of Ghana http://ugspace.ug.edu.gh 2. 6 Acquired immunity 2. 6. 1 Humoral immune response Malaria infection induces strong humoral immune responses through the development of high concentrations of immunoglobulins (Ig), especially IgG and IgM, as well as IgE. The extent of defense against malaria infection in humans and mice has been shown to be correlated with the level of antibodies against asexual blood stage antigens (Piper, 1999; Hirunpetcharat, 1998; Astagneau, 1995). The role of antibodies in malaria immunity is evident in the protection provided to neonates and infants by malaria-specific antibodies that mothers acquire (Sabchareon, 1991; McGregor, 1964). Passive transfer of monoclonal antibodies to plasmodium parasite antigens provided protection to naive mice (Spencer, 1998; Narum, 2000). Although various immunoglobulins protect people from malaria, IgG is the most important. The use of purified immunoglobulin from sera of African adults in clinical trials to treat some sick children substantially decreased clinical symptoms and parasitemia (Bouharoun-Tayoun, 1990). It has been identified that immunoglobulin G (IgG) is the main component of defense against the asexual blood stage of P. falciparum (Druilhe, 1994). 2. 6. 2 Mechanism of antibody response Antibodies are believed to mediate malaria defense through a series of mechanisms. In vivo studies indicate that one mechanism can confer clinical immunity to malaria by antibody interrupting parasite multiplication (McGregor, 1964; Sabchareon, 1991). Antibodies to blood-stage merozoite antigens can block or make parasite invasion of erythrocytes susceptible to phagocytosis, leading to a reduction in parasitemia (Blackman, 1994; Holder, 1992). Other mechanisms are the clearance of infected erythrocytes from circulation by antibodies binding to their surface through Fc receptors and their removal from the body. 14 University of Ghana http://ugspace.ug.edu.gh Some parasites induce antibodies that form clumps or rosettes that the immune system recognizes and eliminates from circulation by opsonization or phagocytosis (Treutiger, 1992). Cell-dependent antibody-mediated cytotoxicity of parasites may be induced by cytophilic antibodies and by inhibition of parasites by effector cells such as neutrophils and monocytes (Bouharoun-Tayoun, 1990). Parasite agglutination and indirect effects such as antibody-dependent cellular inhibition (Oeuvray, 1994) are other immune mechanisms that protect against P. falciparum malaria. Malaria parasites also set up systems for immune evasion. These processes include antigenic variability since many different parasite antigens are introduced to the immune system. Others include modulation of the host immune response that could lead to pathological changes, polymorphism of the parasite protein, and competition between protective and non-protective reactions (Troye- Blomberg, 1984). 2. 6. 3 Cell-mediated immunity Though antibody plays a significant role in malaria immunity, T-cells and cytokines are also involved in immune regulation and effector phases of anti-malaria immunity via T helper cells (Weidanz, 1988). Cell-mediated immune responses to malaria protect against erythrocytic and erythrocytic parasites (Troye-Blomberg, 1984). The importance of cytokines in conferring protection immunity to malaria infection in animal models has been documented (Kobayash, 1996; Shear., 1990; Stevenson, 1990). Several studies have identified cellular processes such as lymphocyte proliferation, IFNy development, activation, and killing of macrophages when peripheral blood mononuclear cells (PBMCs) from immune individuals have been stimulated with malaria antigens in vitro (Ballet, 1985; Brown, 1986; Ockenhouse., 1984) . 15 University of Ghana http://ugspace.ug.edu.gh Similar studies were conducted on non-immune individuals infected with P. falciparum, and decreased cellular recognition of plasmodial antigens was reported (Ho, 1995). T-cells that control the production of antibodies are also involved in inflammation and cytokine regulation; separating TH-cells into their ThI and Th2 subsets may have significant biological and immunological consequences for susceptibility or resistance to diseases or infections (Troye-Blomberg., 1984). Thus, different subsets of Th cells play different roles in inflammation or anti-inflammation in malaria infection. 2. 6. 4 Mechanisms of cellular immune response Studies have shown that ThI and Th2 cells are responsible for regulating immune-mediated antibodies and cell-mediated immunity. This is evident from a study in which T-cells regulated cytokine-induced antibody development through ThI and Th2 (Weidanz, 1988). ThI cells secrete cytokines such as interleukin-2 (IL-2), interferon (IFN) y, tumor growth factor (TGF), and tumor necrosis factor α (TNF α). These cytokines activate macrophages that aid with IgG antibodies, facilitate inflammatory reactions leading to tissue injury, and mediate delayed hypersensitivity reactions (Abbas, 1996). Th2 cells secrete cytokines like IL-4, IL-5, IL-6, IL-10, and IL-13. These cytokines stimulate the proliferation of mast cells and eosinophils and aid B-cells during infection (Abbas, 1996). These cells cross-regulate the differentiation and activity of each other via the cytokines they generate. Studies have shown that Th2-secreted cytokines regulate functional activity and the development of ThI secreted cytokines in P. falciparum infection (Ho, 1995). ThI secreted cytokines regulate the production of Th2 cytokines, while Th2 cells also secrete cytokines that control the production of ThI cells (Troye-Blomberg., 1984). For example, IFNℽ secreted by ThI cells inhibits the development and spread of Th2 cells, while IL-4 16 University of Ghana http://ugspace.ug.edu.gh and IL-10 secreted by Th2 cells inhibit the development of ThI cells. Cytokines such as IL-1, IL- 6, IFN-ℽ, and TNF-α have been shown to defend by inducing macrophages and neutrophils to destroy parasites (Kumaratilake, 1992; Taylor-Robinson, 1993; Troye-Blomberg ., 1999). 2. 7 Haemoglobin variants restrict P. falciparum growth in RBCs Numerous investigations into the invasion and growth of P. falciparum in RBCs containing variant haemoglobin quickly followed the development of in vitro cultivation systems (Trager., 1976; Haynes., 1976). Reductions in RBC invasion have been reported for a variety of haemoglobinopathies, including α-thalassemia trait (Bunyaratvej, 1992), HbH disease (Ifediba, 1985; Chotivanich, 2002), HbEE (Bunyaratvej, 1992; Chotivanich, 2002) HbAE (Chotivanich, 2002) and the compound heterozygous β-thalassemia/HbE disorder (Chotivanich, 2002; Bunyaratvej, 1992; Brockelman, 1987); reductions in the intraerythrocytic growth or maturation of parasites have been reported for HbH disease (Ifediba, 1985; Brockelman, 1987),β-thalassemia minor (Brockelman, 1987) HbSS (Pasvol, 1978; Pasvol., 1980), HbAS (Pasvol, 1978)HbCC (Friedman, 1979; Fairhurst, 2003; Olson, 1986). A recent study proposes a novel mechanism for inhibiting P. falciparum growth in HbS-containing RBCs. Both HbAS and HbSS RBCs display host microRNA (miRNA) profiles that are distinct from those of HbAA RBCs (Chen, 1998; Sangokoya, 2010). 2. 8 Haemoglobin variants interference with mechanisms of P. falciparum Malaria Two major pathogenic iRBC phenotypes were described: those mediating binding of iRBCs to endothelial receptors (Baruch, 1996) and those mediating binding of iRBCs to uninfected RBCs (Carlson, 1990; Kaul, 1991). Both adherence phenotypes are conferred by Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expression (Su, 1995; Baruch, 1996; Smith, 1995). 17 University of Ghana http://ugspace.ug.edu.gh Other pathogenic mechanisms that could be associated with the disease include the production of cytokines in response to P. falciparum glycosylphosphatidylinositol (PfGPI) (Schofield, 1993) and parasite-derived uric acid (Lopera-Mesa, 2012) direct haemolysis due to parasite egress from RBCs. Several studies indicate that the deterioration of cytoadherence interactions partly controls haemoglobinopathy malaria defense. Further analysis of this phenomenon showed that HbAC and HbCC significantly interfered with the binding of iRBCs to human microvascular endothelial cells (HMVECs) under static and physiological flow conditions (Fairhurst., 2005). Subsequent investigations also found significant reductions in the binding of HbAS iRBCs (Cholera, 2008), HbF-containing iRBCs (Amaratunga., 2011), and α-thalassemic iRBCs (Krause, 2012) to human microvascular endothelial cells. Recent research confirms this malaria-protective candidate mechanism; researchers investigated the iRBC's protein-trafficking network and showed that the parasite remodels the actin cytoskeleton of the RBC to export parasite-derived proteins to the iRBC surface knobs (Cyrklaff, 2011). This actin cytoskeleton is disrupted in HbSC and HbCC iRBCs, and the export of parasite proteins to surface knobs is likely to inhibit actin polymerization by hemichromes (Cyrklaff, 2011). These oxidized, denatured haemoglobin accumulate in RBCs containing HbS and HbC, thus providing a possible connection between haemoglobin instability and abnormal PfEMP1/knob display (Cyrklaff, 2011). 2. 9 Haemoglobin variants influence the Innate host defense responses to P. falciparum. The impact of aberrant host responses on the pathogenesis of malaria, especially severe falciparum malaria, is increasingly recognized (Schofield., 2005; Hunt, 2003; Clark, 2004). The innate host defense response encompasses myriad stereotypical pathways that are activated by 18 University of Ghana http://ugspace.ug.edu.gh microorganisms and orchestrated to mitigate insults while minimizing collateral toxicity (Takeuchi, 2010). Typically initiated by recognizing pathogen-associated molecular patterns (PAMPs) by leukocyte-associated Toll-like Receptors (TLRs), these responses then progress through: 1. A pro-inflammatory phase is marked by the release of cytokines, activation of endothelial cells, and recruitment of circulating and locally resident immune effector cells. 2. A counter-regulatory phase in which tissue-protective molecules such as erythropoietin (Villa, 2003; Siren, 2001), heme oxygenase-1 (HO-1) (Wagener, 2001; Kapturczak, 2004), and angiopoietin-1 (Kim, 2011) are deployed to limit inflammatory damage. 3. A repair phase is mediated by vascular- and tissue-specific stem cells (Koh, 2009; Erbayraktar, 2009). Such phases emerge from the host and pathogenic factors that jointly regulate specific responses that are pro-inflammatory and counter regulatory. In P. falciparum malaria, these innate immune responses are potently triggered by PfGPI (Krishnegowda, 2005) and haemozoin (Parroche, 2007; Jaramillo, 2004) activation of TLRs on leukocytes, as well as by microvascular inflammation induced by PfEMP1-mediated binding of iRBCs to the endothelium (Tripathi, 2009). Severe malaria has been associated with increased angiopoietin-2, decreased angiopoietin-1, and decreased levels of endothelial nitric oxide, and counter-regulatory molecules including HO-1 and erythropoietin have been identified in human studies (Taylor, 2013). The association of HO-1 levels with severe malaria was variable in Gambian children and HO-1 promoter polymorphisms which confer higher constitutive levels of 19 University of Ghana http://ugspace.ug.edu.gh HO-1 was associated with an increased risk of severe malaria (Taylor, 2013). Such results, obtained mainly from HbAA patients, indicate that HO-1 can be either protective or deleterious over a wide range of in vivo rates (Taylor, 2013). Given the lack of studies on haemoglobinopathies and innate host defenses, a separate line of nitric oxide (NO) and extreme malaria investigations highlight the significance of interactions between iRBC and endothelium in serious malaria pathogenesis. NO also exhibits anti-inflammatory behavior by reducing the expression of host receptors used by iRBCs to bind microvascular endothelial cells (De-Caterina, 1995). Thus, haemoglobinopathies and increased NO production are associated with in vivo defense against severe malaria and decreased binding of iRBCs to in vitro endothelium. 2. 10 Haemoglobin variants enhance the adaptive immune responses to P. falciparum Field studies evidence suggests an association between several haemoglobinopathies, adaptive immunity, and malaria defense, while investigations of these relationships are complicated by the lack of accurate correlates of immune response (Taylor, 2013). The ability of polyclonal IgGs from malaria-immune adults to clear parasitemia in children with malaria indicates a central role for antibodies in malaria immunity (Cohen, 1961). Antibody-mediated phagocytosis of iRBCs is an essential mechanism of effectors in malaria defense; research with normal RBCs has shown that monocytes prefer iRBCs to phagocytosis compared to uninfected RBCs and that this preference is potentiated by binding IgG to iRBCs (Celada, 1982). In addition, hyper-immune sera polyclonal IgG binds more avidly to both α-thalassemic iRBCs (Luzzi, 1991; Williams., 2002) and β- thalassemic iRBCs (Luzzi, 1991) compared to non-thalassemic iRBCs, indicating that this mechanism can ideally clear iRBCs with haemoglobin variants. While these data suggest that Haemoglobinopathies functionally enhance the clearance of iRBCs, the precise mechanism of this enhancement remains unclear. There is no evidence of this role in the clearance of parasites by other 20 University of Ghana http://ugspace.ug.edu.gh haemoglobinopathies and the correlation between this mechanism and the protection from the clinical disease have not been specifically investigated. Attenuating host-cell injury, coupled with mechanisms of parasite growth restriction in haemoglobinopathic iRBCs, may be involved in prolonging the asymptomatic phase of parasitemia. This delay in developing symptoms may provide more time for erythrocytic stage antigens and RBC senescence markers to be exposed to the immune system, thus enhancing both the acquisition and maintenance of adaptive and memory- based immune responses that ultimately protect individuals from developing the deadliest complications of P. falciparum infection. 2.11 Malaria Diagnostics Malaria presents a diagnostic challenge to laboratories in most countries. The urgency and importance of obtaining results quickly from examining blood samples from patients with suspected acute malaria render some of the more sensitive methods for malaria diagnosis impractical for routine laboratory use (Moody, 2002). Changing patterns of known morphological appearances of malaria species, possibly due to drug pressure, strain variation, or approaches to blood collection, have created diagnostic problems that cannot easily be resolved merely by reference to an atlas of parasitology (Moody, 2002). Thus, one of the primary interventions of the Global Malaria Control Strategy by the WHO is prompt and accurate diagnosis of the disease (WHO,1999) 21 University of Ghana http://ugspace.ug.edu.gh 2.12 Malaria serology Malaria endemicity has been assessed using antibody prevalence determined by immunofluorescence (Voller & O’Neill, 1971 & Druilhe et al., 1986). Still, its use was limited by reliance on cultured parasites, expensive fluorescence microscopes, and the subjective nature of slide reading. Subsequently, the measurement of antimalarial antibodies by ELISA was shown to be a potentially helpful epidemiological tool (Esposito, 1988). The resistance of P. falciparum threatens malaria elimination efforts to anti- malarial drugs by increasing and widespread mosquito vector resistance to insecticides and by the lack of an effective vaccine conferring strong protective immunity to infection. In malaria-endemic areas, human populations develop natural immunity against P. falciparum which can lead to premonition (Crompton, 2010; Lu, 2017; Ranson, 2016). This acquired protective immunity takes years to establish following recurrent Plasmodium parasite exposure, is relatively short-lived, and is only partially effective. It can efficiently control malaria parasite infection leading to a decline in clinical malaria since low parasitemia mainly persists in the presence of circulating antibodies (Abs). Specific Abs, such as immunoglobulin G, primarily mediate protective immunity. Immune responses are complex traits, and vaccine development necessitates extensive information on human populations' processes and determinants that modulate immune responses. The effect of age, genetic factors, pathogen co-infection, and nutritional status have been more intensively explored and are recognized to influence anti-Plasmodium Ab responses and to have some association with malaria clinical protection. Antibodies are essential to the acquisition of defense against malaria. During the developmental phases of P. falciparum, the parasites transmit specific proteins or antigens on their surfaces. Antibodies to these antigens provide protection through several mechanisms (Groux, 1990). Some of the antigens found on P. falciparum include Erythrocyte binding antigen175 (EBA-175), Gamete surface antigen, Pfs230, Merozoite surface 22 University of Ghana http://ugspace.ug.edu.gh protein 3 (MSP3), the Circumsporozoite Surface Protein (CSP), P. falciparum Erythrocyte Membrane Protein (PfEMPl), Apical Merozoite Antigen 1 (AMA1), Liver stage antigen (LSA), and the Glutamate Rich Protein (GLURP). In recent immuno-epidemiological studies, several antigens have been identified as possible targets for anti-malaria defense. Which include GLURP, MSP1-19, and MSP3, which are currently in phase I clinical trials CSP (Hoffman, 1992), LSA, and PfEMPl (Bull., 2016) have also been studied in connection with protection. 2.12.1 Asexual Stage Antigen - PfEBA-175 PfEBA-175 has been shown to play a crucial role during the fast cascade of interactions between the parasite and host molecules before the merozoites ultimately invade the erythrocyte (Chen, 2013). PfEBA-175 binds to glycophorin A on the surface of erythrocytes, and this interaction depends on Salic acid on the receptor (Sim, 1994). Antibodies induced against diverse antigenic components of the erythrocytic parasite are essential mediators of anti-disease immunity (Amoah, 2018). Antibodies specific to EBA 175 RIII-V are associated with protection from malaria in symptomatic cases (Amoah, 2018). In the absence of infection, however, a few investigations, notably by Wipasa et al., found that antibody and memory B cell responses to malaria antigens remained constant throughout time (Wipasa, 2010). This antigen was chosen because it’s a potential vaccine candidate and uses different invasion pathways. During the asexual erythrocytic phase of the P. falciparum life cycle, the parasite population grows exponentially, resulting in clinical symptoms. The rapidity with which merozoites invade erythrocytes suggests that particular receptor-ligand interactions between host and parasite molecules are involved (Duraisingh, 2003) And because erythrocyte invasion is an obligate part of the parasite’s lifecycle, blocking invasion should prevent parasite growth, and this has become 23 University of Ghana http://ugspace.ug.edu.gh an attractive target for vaccine development. P. falciparum invades erythrocytes by using multiple receptor-ligand interactions defined as invasion pathways (Camus, 1985; Doolan, 1999). The first P. falciparum ligand identified to bind to erythrocytes with high affinity was erythrocyte- binding antigen 175 (Camus, 1985). Before the merozoite completely invades the erythrocyte, PfEBA-175 has been demonstrated to play an essential part in the quick cascade of interactions between parasites and host molecules (Aikawa, 1978). PfEBA-175 binds to glycophorin A on the surface of erythrocytes, and this interaction depends on sialic acid on the receptor (Sim, 1994). The binding region of PfEBA-175 to glycophorin A involves the cysteine-rich region II consisting of two halves called F1 and F2 domains. Antibodies raised against the F2 domain of PfEBA-175 have been shown to partially inhibit the invasion of P. falciparum merozoites into human erythrocytes (Narum, 2000). Antibodies recognize PfEBA-175 RII in individuals with naturally acquired immunity (Okenu, 2000). In addition, antibody levels are associated with protection from malaria (Okenu, 2000; McCarra, 2011; Richards, 2008), although this association is not observed in groups with a low incidence of disease (Osier, 2014) 2.12.2 Sexual Stage Antigen - Pfs230 Pfs230 is a surface antigen of P. falciparum that has shown transmission-blocking activity. Transmission-blocking immunity to P. falciparum in malaria-immune individuals was associated with antibodies to the gametocyte surface protein Pfs230 (Healer, 1999). Their results confirm Pfs230 as a possible candidate for inclusion as a malaria transmission-blocking vaccine. A previous study by (Eksi., 2002) demonstrated that targeted disruption of Pfs230 resulted in the production of truncated proteins that were not retained on the surface of the gametocyte or gamete (Eksi, 2006). Pfs230 disruptants successfully emerge from RBCs and male gamete sex flagellate-producing microgametes (Eksi, 2006). However, ex-flagellating Pfs230- minus males could not 24 University of Ghana http://ugspace.ug.edu.gh Interact with RBCs and form exflagellation centers. Oocyst production and mosquito infectivity were also significantly reduced, 96–92% and 76–71%, respectively (Eksi, 2006). Their results suggest that Pfs230 is a surface molecule in males that mediates RBC binding and plays an essential role in oocyst development, a critical step in malaria transmission (Eksi, 2006). In a recent study conducted by (Farrance, 2011), they successfully produced a region of the Pfs230 antigen in their plant-based transient-expression system and evaluated the role of the protein in an animal model. In rabbits with high titers of transmission-blocking antibodies, administration of 230CMB with > 90 % purity causes robust immunological responses, resulting in a greater than 99 % reduction in oocyst counts in the presence of complement, as assessed by a conventional membrane feeding assay (Farrance, 2011). Additionally, sera from people with a strong Pfs230 response have been demonstrated to have transmission-reducing immunity. Several studies looking at naturally acquired immune responses to Pfs230 have now been conducted across several sites to expand our understanding of naturally acquired immunity to sexual stage antigens. To understand the dynamics of sexual stage immunity, traditional indices of parasite exposure, such as age, transmission setting, malaria transmission season, and parasite prevalence, have been analyzed. However, the reported relationships between the parameters above and the seroprevalence of antibodies to sexual-stage antigens are inconsistent. Immune responses to Pfs230 were studied in ten research spanning 15 study sites in Africa. The range of seroprevalence estimates was quite broad, ranging from 6% reported by Stone et al. in Soumousso and Dande villages, Burkina Faso, to 72% reported by (Amoah, 2018). Immunological responses to numerous malaria parasite antigens should be studied, and persons with haemoglobinopathies should be included in these studies. As a result, this study will shed further information on how changes in haemoglobin morphologies in symptomatic malaria patients affect natural malaria protection (IgG levels). 25 University of Ghana http://ugspace.ug.edu.gh 2.12.3 Salivary Gland antigen- gSG6-P1 Human antibodies recognizing the Anopheles gambiae salivary protein gSG6 P1 were associated with recent exposure to Anopheles bites in tropical Africa. These observations suggested that anti-gSG6-P1 antibody levels might serve as an entomological proxy to estimate anopheles biting intensity by reflecting recent exposure to anopheles bites (Pollard, 2016). In the case of malaria, a recent study has shown that human IgG response to the gSG6-P1 peptide represented a specific biomarker of mosquito bites' exposure, which will aid malaria surveillance. This will better understand the influence of haemoglobin variants on IgG levels in symptomatic malaria in Ghana, thus correlating the IgG levels to haemoglobin variants across Ghana. So far, there’s minimal information on the influence of haemoglobin variants such as HbAS and HbAC on Pfs230, EBA 175 3R, and gGS6-P1 levels in symptomatic patients across Ghana. 2.13 Capillary electrophoresis The Capillarys haemoglobin (E) kit is designed for the separation of the normal haemoglobins (A, A2, and F) in human blood samples, and for the detection of the significant haemoglobin variants (S, C, E, and D), by capillary electrophoresis in alkaline buffer (pH 9.4) with the SEBIA capillarys two flex-piercing instruments. The capillarys haemoglobin (E) kit is designed for laboratory use. The capillarys two flex-piercing instruments is an automated analyzer that performs a complete haemoglobin profile for the quantitative analysis of the normal haemoglobin fractions A, A2, and F and the detection of significant haemoglobin variants S, C, E, and D. The assay is performed on the haemolysate of whole blood samples collected in tubes containing K2EDTA or K3EDTA as an anticoagulant 26 University of Ghana http://ugspace.ug.edu.gh Principle of the test Haemoglobin is a complex molecule composed of two pairs of polypeptide chains. Each chain is linked to the haem, a tetrapyrrolic nucleus (porphyrin) that chelates an iron atom. The haem part is common to all haemoglobin and their variants. The type of haemoglobin is determined by the protein part called globin. Polypeptide chains α, ß, δ and γ constitute the normal human haemoglobins: • haemoglobin A .............................................. = α2 ß2 • haemoglobin A2 ............................................ = α2 δ2 • fetal haemoglobin F ...................................... = α2 γ2 The α-chain is common to these three haemoglobins. The haemoglobin spatial structure and other molecular properties (like all proteins) depend on the amino acids' nature and sequence constituting the chains. Substitution of amino acids by mutation is responsible for forming haemoglobin variants with different surface charges and electrophoretic mobilities, which also depend on the pH and ionic strength of the buffer. The resulting qualitative (or structural) abnormalities are called haemoglobinopathies (Landers, 1995; Livingstone, 1986; Schneider, 1978). Decreased synthesis of one of the haemoglobin chains leads to quantitative (or regulation) abnormalities called thalassemias. Haemoglobin electrophoresis is a well-established technique routinely used in clinical laboratories for screening samples for haemoglobin abnormalities (Bardakdjian-Michau, 2003; Fairbanks, 1980; Galacteros, 1986; Hempe, 1997; Oda, 1997). The capillarys two flex-piercing instruments have been developed to provide complete automation of this testing with fast separation and reasonable resolution. The methodology can be considered an intermediary between classical zone electrophoresis and liquid chromatography (Krauss, 1986; Maier-Redelsberger, 1989). The capillarys two flex-piercing instrument uses the principle of capillary electrophoresis in free 27 University of Ghana http://ugspace.ug.edu.gh solution. This technique separates charged molecules by electrophoretic mobility in an alkaline buffer with a specific pH. Separation also occurs according to the electrolyte pH and electroosmotic flow (Huisman, 1977). The capillary's two flex-piercing instruments have capillaries functioning in parallel, allowing eight simultaneous analyses for haemoglobin quantification from whole blood samples. A sample dilution with haemolysing solution is prepared and injected by aspiration at the anodic end of the capillary. A high voltage protein separation is then performed, and the haemoglobins are directly detected at 415 nm at the cathodic end of the capillary. Before each run, the capillaries are washed with a wash solution and prepared for the subsequent analysis with buffer. The haemoglobins, separated in silica capillaries, are directly and detected explicitly at an absorbance wavelength of 415 nm, which is specific to haemoglobins. The resulting electropherograms are evaluated visually for pattern abnormalities. Direct detection provides accurate relative quantification of individual haemoglobin fractions, with particular interest, such as A2 haemoglobin for ß thalassemia diagnostic. In addition, the high resolution of this procedure should allow the identification of haemoglobin variants, in particular, to differentiate haemoglobins S from D and E from C. The haemoglobin A2 quantification can also be performed when haemoglobin E is present. By using an alkaline pH buffer, normal and abnormal (or variant), haemoglobins are detected in the following order, from cathode to anode: δA’2 (A2 variant), C, A2/O-Arab, E, S, D, G-Philadelphia, F, A, Hope, Bart’s, J, N-Baltimore, and H. The carbonic anhydrase is not visualized on the haemoglobin electrophoretic patterns; this permits the identification of haemoglobin A2 variants in this migration zone. 28 University of Ghana http://ugspace.ug.edu.gh Figure 2: Capillary electrophoresis system; CAPILLARYS 2 FLEX PIERCING Figure 3: A typical separation profile obtained with the Capillarys 2 Flex Piercing analyzer. 29 University of Ghana http://ugspace.ug.edu.gh 2.14 Enzyme-Linked Immunosorbent Assay (ELISA) ELISA is a common laboratory technique used to measure an analyte's concentration (usually antibodies or antigens) in a solution. ELISA begins with a coating step, where the first layer- either an antigen or antibody is adsorbed (passive attachment of a liquid to a solid surface, creating a thin film) to a solid surface, usually a polystyrene multiwell plate. The coating is followed by blocking and detection steps. When there is interaction, this can be detected by a conjugate (an anti-human Ig coupled to an enzyme linked to a substrate), which reduces the substrate to produce a visible color change. The colored end product correlates to the amount of analyte in the original sample. ELISA is a very useful tool in malaria research, where it has been used to diagnose tertian malaria (Ming, 1987) and for determining Plasmodium falciparum antigens (Spencer et al., 1979). There are three different types of ELISA based on the detection method. They are Direct, Indirect, and Sandwich/Capture assays. The difference between Direct ELISA and Indirect ELISA is that Direct ELISA involves labeling the detection antibody with an enzyme or an alternative signaling molecule such as a fluorophore. In contrast, Indirect detection involves an additional probe (a secondary antibody) labeled with a detectable tag. This secondary antibody produces a measurable signal tag by binding to the detection (primary) antibodies. Direct ELISA is faster than Indirect ELISA but is less sensitive and cannot provide the amplification gained from using a secondary antibody. With Sandwich ELISA, the antigen is quantified between two layers of antibodies (i.e., a capture and detection antibody). To detect antigen, wells of microtiter plates are coated with capture antibodies (usually monoclonal or polyclonal antibodies) followed by incubation with the test solutions containing the antigen. Afterward, an antigen-specific antibody conjugated to an enzyme is applied as the detection antibody. This is a direct sandwich ELISA. But there is also Indirect Sandwich ELISA where an antigen-specific detection antibody is used to sandwich the incubated antigen before introducing the enzyme-linked secondary antibody for detection. 30 University of Ghana http://ugspace.ug.edu.gh CHAPTER 3 MATERIALS AND METHODS 3.1 Study type and description of study sites This is a nested study to compare IgG levels of malaria parasite and vector antigens in two populations and determine the frequency of different haemoglobin variants in the microscopy-positive malaria population. Ten health facilities in Ghana's previous ten regions were chosen randomly. The facility size was calculated using Probability proportional to size estimates (PPSE) based on the total monthly number of confirmed malaria cases in outpatient departments (OPDs) in 2016. Three district/municipal hospitals and seven health centers were selected from each of the ten Regions(New Abirem Hospital, Shai-Osukodu Hospital, Abura Dunkwa District Hospital, Praso Health Centre, Gyedu Health Centre, Kalba Health Centre, Paga Health Centre, Lambussi Polyclinic, Kpetoe Health Centre and Agona Nkwanta Health Centre). The research included any patient suspected of uncomplicated malaria at the designated health. 3.1.1 Subjects/study population  Archived packed cells and plasma collected from suspected malaria patients seeking treatment in all the participating health facilities that have been consented for reuse 3.2 Inclusion Criteria  All samples used in this study were archived samples collected within 2018-2019 and aged 2 to 89 years. 3.3 Exclusion Criteria  Archived samples containing any other species of Plasmodium aside Falciparum was excluded from the study 31 3.4 Sample size determination The sample size was determined using the binomial model to estimate the confidence interval (CI) (Vallejo, 2013). The sample size with a 95% CI and precision level of 5% was estimated according to the formulae below: The sample size was calculated with the formula below. <2 Û2:1 F2; N = A2 Where N = sample size, Z = statistic corresponding to the level of confidence, P = expected prevalence and e = precision.  With a level of confidence at 95% corresponding to Z = 1.96  The proposed prevalence of malaria diagnosis by microscopy is 25.2 % (Kweku, 2017)  Precision set at 5% The minimum number of samples based on the above calculation was 288; thus, 300 microscopy-positive archived samples and 300 microscopy-negative archived samples were used for the study. 32 University of Ghana http://ugspace.ug.edu.gh 3.5 Ethical Consideration Ethical clearance was obtained from the Institutional Review Board (IRB) of Noguchi Memorial Institute for Medical Research (NMIMR) and the Ethical Committee of the College of Health Sciences of the University of Ghana. 3.6 MATERIALS 3.6.1 Reagents Deep well plates, plastic seals, plate reader: Bio-Rad Model 680 Microplate Reader, plate washer, Tween-20, absolute ethanol, sulphuric acid were all obtained from Sigma-Aldrich (U.S.A), MAXISORP NUNC-immune plates from Thermo Scientific, Anti-Human Immunoglobulin G (H+L) HRPO conjugate from Life Technology, Phosphate buffered saline from Oxoid, (England) and dried skimmed milk from Marvel, (Ireland), Haemolysing solution, Capillarys 2 flex piercing instrument. 3.7 METHODS 3.7.1 Examination of malaria Parasite by Microscopy The archived samples used for this research were previously analyzed for the presence of malaria parasites by two independent WHO-certified malaria microscopists; any disagreement on smear (thick and thin) readings was resolved by re-examination by a third microscopist’s assessment. The assessment by the third microscopists was considered to be the final decision. 33 University of Ghana http://ugspace.ug.edu.gh 3.7.2 Indirect ELISA Enzyme-linked immunosorbent assay (ELISA) was used to determine the natural levels of antibodies of the Salivary Gland peptide (gSG6-P1), asexual stage antigen (PfEBA-175 Region III), and sexual stage antigen (Pfs230). NUNC 96-well streptavidin-coated ELISA plates were coated with 100 µl of 1 µg/ml of Mosquito Salivary Gland antigen in phosphate-buffered saline (PBS, pH 7.4). Also, NUNC 96-well ELISA plates were coated with 20 µl of 1 µg/ml of asexual stage antigen in phosphate-buffered saline (PBS, pH 7.4) and 100 µl of 1 µg/ml of sexual stage antigens in carbonate buffer (0.05 M carbonate/bicarbonate buffer, pH 9.2) and kept overnight at four °C. The plates were washed four times with wash buffer (1X PBS and 0.0 5% Tween- 20). Each washing step involved emptying the contents of the wells of unbound materials by flipping the plate, adding 250 µl of wash buffer, and incubating for 1 min. The wash buffer is then discarded. Unbound regions in the wells were then blocked by incubating with 150 µl of blocking buffer (3% skimmed milk in wash buffer) for 1 hr. The plates were then washed twice with wash buffer as before and incubated for 1 hour with 100 µl per well of 1:200 dilutions (samples) and 1% skimmed milk for dissolving negative and positive controls consisting of pools of previously sampled high titer plasma. Plates were washed as before, and wells incubated with 50 µl of peroxidase-conjugated antibodies (1:3000 dilution of goat antihuman IgG-HRPO) for 1 hr. The plates were washed and incubated with incubated peroxidase substrate TMB (3,3',5,5'- Tetramethylbenzidin) for 30 min, and the enzyme The reactions were stopped by adding 50 µl of 0.2 mM sulphuric acid. Care was taken to ensure that all plates were incubated with a substrate for relatively the same time. The optical densities of the contents of the wells were then read using the Biotek ELISA plate reader at 450 nm. 34 University of Ghana http://ugspace.ug.edu.gh The reader determined the optical densities of the contents of each well using the Beer-Lambert Law. Briefly, the distribution of log10-transformed OD values was fitted as the sum of two Gaussian distributions using maximum likelihood methods; one of the two distributions represents the seronegative population, and the other represents the seropositive population. For each antigen, the mean log10-OD of the Gaussian corresponding to the seronegative population plus log10-three standard deviations was calculated, back-transformed, and used as the cut-off for seropositivity. Seroprevalence was calculated as the proportion of samples with OD above this cut-off. 3.7.3 Haemoglobin Phenotype determination Capillary electrophoresis was performed on symptomatic samples; Samples were brought to thaw at room temperature. The samples were then arranged in sample tubes in racks according to sample ID order. A working list was prepared per the sample ID order. Then, 200 µL of the haemolysing solution was pipetted into Eppendorf tubes labeled according to the sample ID order. The samples were vortexed, and 25 µL of the sample was pipetted into the Eppendorf tubes containing the haemolysing solution. The mixture was then vortexed for 5 seconds. The dilution segment with the capillary two flex piercing equipment was placed on the sample rack. 100 µL of the haemolysate was pipetted into the dilution segment from 1 to 8. The sample rack was slithered into the Capillarys 2 Flex-Piercing instrument to start the analysis. 3.8 Data analysis The ADAMSEL software was used to convert OD values obtained for the samples into weighted concentration values. Log-transformed antibody data were analyzed using SPSS (version 16.0) and GraphPad Prism (GraphPad Software, version 5.01). A Mann-Whitney U test (a nonparametric test used to replace an unpaired T-test) was used to compare means between positive and negative malaria microscopy patients. Kruskal Wallis Variational Test was performed to compare the different IgGs of the various haemoglobin variants. 35 University of Ghana http://ugspace.ug.edu.gh CHAPTER 4 RESULTS A total of 600 archived samples from malaria suspected patients in the ten regions of Ghana were used in this study. Out of the samples collected, 344 were from females, whiles 256 were from males. The average age across the ten regions was 21.84 years, while the minimum and maximum ages recorded were 1 year and 88 years, respectively, as shown in Table 1 Table 1: Demographic Characteristics of the Study Participants Grouped Regionally. Parameter A BA C E GA N UE UW V W TOTAL (60) (60) (60) (60) (60) (60) (60) (60) (60) (60) SEX MALE 25 33 27 23 26 29 20 24 25 24 256 FEMALE 35 27 33 37 34 31 40 36 35 36 344 AGE (YRS) MEAN 20.57 19.40 23.98 19.75 27.75 22.85 22.90 21.15 21.80 18.27 21.84 SEM 2.66 2.50 3.10 2.55 3.58 2.95 2.96 2.73 2.81 2.36 2.82 MINIMUM 2 2 2 3 3 1 1 1 1 1 1.70 MAXIMUM 83 72 83 78 77 81 82 88 85 72 80.40 SEM, standard error of the mean; A, Ashanti; BA, Brong Ahafo; C, Central; E, Eastern; GA, Greater Accra; N, Northern; UE, Upper East; UW, Upper West; V, Volta; W, Western. 36 University of Ghana http://ugspace.ug.edu.gh Objective 1: TO DETERMINE AND COMPARE IGG LEVELS IN THREE MALARIA ANTIGENS IN MALARIA PATIENTS. Table 2: Comparison of the IgG Levels for gSG6-P1 in Malaria Positive and Negative Individuals at Each Region Using Mann Whitney U Test Region Min Max Mean ± SD 95% CI P Ashanti 0.1421 Neg 443.60 5995.00 1513.00±1189.00 1069.00-1958.00 Pos 0.00 2760.00 1503.00±598.80 1279.00-1726.00 Brong Ahafo 0.0013 Neg 750.10 3573.00 2822.00±6260.00 484.50-5159.00 Pos 455.60 2409.00 1141.00±475.30 963.40-1318.00 Central 0.8087 Neg 607.10 4739.00 1709.00±946.20 1356.00±2063.00 Pos 0.00 2690.00 1511.00±610.40 1283.00-1739.00 Eastern 0.2359 Neg 0.00 3364.00 1461.00±661.60 1214.00-1708.00 Pos 0.00 3364.00 1296.00±748.10 1016.00-1575.00 Greater Accra 0.1910 Neg 678.70 5866.00 1652.00±1150.00 1223.00-2081.00 Pos 0.00 2295.00 1147.00-533.10 947.40-1346.00 Northern 0.5325 Neg 459.20 3979.00 1384.00±772.80 1096.00-1673.00 Pos 619.30 3852.00 1506.00±864.50 1183.00-1828.00 Upper East 0.0850 Neg 0.00 3074.00 1398±729.10 1126.00-1670.00 Pos 415.40 2340.00 1129.00±436.10 966.20-1292.00 Upper West 0.0319 Neg 954.80 4643.00 2328.00±917.10 1985.00-2670.00 Pos 0.00 5114.00 1872.00±1176.00 1433.00-2311.00 Volta 0.0009 Neg 0.00 2947.00 1237.00±622.90 1004.00-1469.00 Pos 781.20 5697.00 2270.00±1415.00 1741.00-2798.00 Western 0.0446 Neg 754.60 3983.00 1990.00±771.80 1702.00-2278.00 Pos 627.50 4936.00 1686.00±1057.00 1292.00-2081.00 Pos; Positive malaria microscopy sample, Neg; Negative malaria microscopy samples, Min; Minimum, Max; Maximum, Mean ± SD; Mean ± Standard Deviation, 95%CI; 95% Confidence Interval, P<0.05 implies statistically significant. A Mann-Whitney U test (a non-parametric test that is used to replace an unpaired T-test) was used to compare the IgG levels for gSG6-P1 in the positive and negative samples collected from each 37 University of Ghana http://ugspace.ug.edu.gh Region. The minimum, maximum, and mean standard deviations of positive and negative malaria samples from each region were compared. For instance, the minimum levels of the IgG for gSG6- P1 for malaria negative and positive samples from the Ashanti region were 443.60 and 0.00, respectively. An example of the maximum concentration of IgG levels for gSG6-P1 observed were 4739.00 and 2690.00 in microscopy negative and positive samples, respectively, from the Central Region. The Brong Ahafo, Upper West, and Western regions observed significantly (p<0.05) high levels of the IgG for gSG6-P1 for malaria negative samples when the means were compared to that of the positive malaria microscopy samples. However, samples from the Volta region recorded an average of significantly higher IgG levels for gSG6-P1 for malaria microscopy positives than for malaria microscopy negatives. The mean comparison of the IgG levels for gSG6-P1 for all other regions insignificantly indicated that the majority of the regions recorded higher levels of the IgG in malaria microscopy negative samples than positive samples. (Table 2) 38 University of Ghana http://ugspace.ug.edu.gh Table 3: Comparison of the IgG Levels for Pfs-230 in Malaria Positive and Negative Individuals at Each Region Using Mann Whitney U Test. Region Min Max Mean ± SD 95% CI P-value Ashanti <0.0001 Neg 0.00 19705.00 3741.00±4154.00 2190.00±5293.00 Pos 4023.00 31670.00 9617.00±6495.00 7192.00±12043.00 Brong Ahafo <0.0001 Neg 1608.00 10083.00 3746.00±1810.00 3070.00-4422.00 Pos 550.70 6838.00 1607.00±1205.00 1157.00-2057.00 Central 0.1159 Neg 725.40 6550.00 2605.00±1596.00 2009.00-3201.00 Pos 664.20 7236.00 2079.00±1444.00 1540.00±2618.00 Eastern 0.0433 Neg 0.00 12252.00 2538.00±2744.00 1513.00-3562.00 Pos 0.00 5227.00 1400.00±1037.00 1013.00-1788.00 Greater Accra 0.0307 Neg 0.00 4538.00 1279.00±1083.00 874.90-1684.00 Pos 0.00 4099.00 1604.00±820.40 1298.00-1911.00 Northern 0.4147 Neg 874.6 10579.00 2963.00±1824.00 2882.00-3644.00 Pos 0.00 34074.00 4146.00±7222.00 1449.00-6842.00 Upper East 0.4829 Neg 0.00 19817.00 3742.00±3870.00 2297.00-5187.00 Pos 0.00 8597.00 2876.00±2047.00 2112.00-3640.00 Upper West 0.9124 Neg 964.40 6395.00 5246.00±2079.00 994.90-9497.00 Pos 1128.00 7104.00 3098.00±1585.00 2506.00-3690.00 Volta 0.5793 Neg 927.50 15022.00 3463.00±3344.00 2214.00-4712.00 Pos 1205.00 8961.00 2701.00±1595.00 2105.00-3296.00 Western 0.7523 Neg 0.00 15504.00 3429.00±3064.00 2285.00-4573.00 Pos 0.00 6753.00 2900.00±1753.00 2245.00-3555.00 Pos; Positive malaria microscopy sample, Neg; Negative malaria microscopy samples, Min; Minimum, Max; Maximum, mean±SD; Mean ± Standard Deviation, 95%CI; 95% Confidence Interval, P<0.05 implies statistically significant. Table 3 Compares the IgG levels for Pfs-230 in malaria-positive and negative samples in each region using the Mann- Whitney U Test. The minimum, maximum and 39 University of Ghana http://ugspace.ug.edu.gh mean±standard deviation of the IgG levels for Pfs-230 positive and negative malaria samples from each region was compared. For example, the minimum levels of the IgG for Pfs-230 for malaria negative and positive samples from the Upper West region were 964.40 and 1128.00, respectively. Regarding the maximum concentrations, the IgG levels for Pfs-230 observed in microscopy negative and positive samples from the Northern region were 10579.00 and 34074.00, respectively. On average, there was a significant rise in the IgG levels for the malaria negative samples (3746.00±1810.00) compared to that of the malaria microscopy positive samples (1607.00±1205.00) in the Brong Ahafo Region. On the other hand, the levels of IgG for Pfs-230 in the malaria microscopy positive samples from the Ashanti region (9617.00±6495.00) and the Greater Accra Region (1604.00±820.40) were significantly higher than the IgG levels for Pfs-230 in negative malaria microscopy samples in the two regions (3741.00±4154.00 and 1279.00±1083.00 respectively). No significant relationships were established between levels of IgG for Pfs-230 in the rest of the regions. However, it is worth noting that although the concentrations observed in the other regions were significant, most of these regions had higher levels of IgG for Pfs-230 in the malaria microscopy negative samples. 40 University of Ghana http://ugspace.ug.edu.gh Table 4: Comparison of the IgG Levels for EBA 175 3R in Malaria Positive and Negative Individuals at Each Region Using Mann Whitney U Test. Parameter Min Max Mean ± SD 95% CI P-value Ashanti 0.0745 Neg 0.00 106964.00 19521.00±24655.00 10314.00-28727.00 Pos 0.00 82864.00 11866.00±19180.00 4704.00-19028.00 Brong Ahafo 0.0358 Neg 3009.00 77800.00 16520.00±20035.00 9039.00±24002.00 Pos 1899.00 75545.00 16520.00±13546.00 4610.00±14727.00 Central 0.797 Neg 860.70 53898.00 9412.00±10392 5531.00±13293.00 Pos 1308.00 37808.00 10309.00±1756.00 6717.00-13901.00 Eastern 0.8545 Neg 1193.00 216558.00 18653.00±40921.00 3372.00-33933.00 Pos 860.70 53898.00 9412.00±1897.00 553100±13293.00 Greater Accra 0.001 Neg 0.00 41847.00 11384.00±10993.00 7279.00-15488.00 Pos 0.00 17293.00 4397.00±863.00 2629.00-6165.00 Northern <0.0001 Neg 0.00 8433172.00 851966.00±2004095.00 103655.00- 1600338 Pos 1354.00 118048.00 10354.00±21383.00 2370.00±18339.00 Upper East 0.4581 Neg 0.00 62019.00 10711.00±14635.00 5246.00±16176.00 Pos 909.80 38258.00 9595.00±9522.00 6039.00-13150.00 Upper West 0.3898 Neg 6226.00 115033.00 16736.00±20696.00 9008.00±24464.00 Pos 0.00 241151.00 33640.00±56509.00 12539.00±54741.00 Volta 0.6650 Neg 0.000 335362.00 30211.00±61626.00 7200.00±53223.00 Pos 2727.00 210490.00 20693.00±38125.00 6456.00±34929.00 Western 0.4125 Neg 0.00 132626.00 16737.00±30062.00 5511.00-27962.00 Pos 1393.00 247994.00 14825.00±44724.00 -1875.00-31526.00 Pos; Positive malaria microscopy sample, Neg; Negative malaria microscopy samples, Min; Minimum, Max; Maximum, mean ± SD; Mean ± Standard Deviation, 95%CI; 95% Confidence Interval, P<0.05 implies statistically significant. Table 4: Compares the IgG levels for EBA 175 3R in malaria microscopy positive and negative samples at each region using the Mann-Whitney U test. The minimum, 41 University of Ghana http://ugspace.ug.edu.gh maximum and mean±standard deviation of the levels of IgG for EBA 175 3R positive and negative malaria microscopy samples from each region were compared with a P<0.05, indicating a statistically significant comparison. The minimum concentrations observed in the negative and positive samples from the Eastern region were 1193.00 and 860.70, respectively. The maximum concentrations of IgG levels for EBA 175 3R in negative and positive samples in the same region were 216558.00 and 53898.00. Notably, the comparison of the IgG levels for EBA 175 3R for all regions was insignificant statistically except in the Brong Ahafo, Northern, and Central Region of Ghana. The IgG levels for EBA 175 3R in the Greater Accra Region were 11384.00±10993.00 and 4397.00±863.00 for the microscopy negative and positive samples, respectively. Table 5: Comparison of the Levels for IgG gSG6-P1, Pfs230, and EBA 175 3R in Malaria Microscopy Positive and Negative Individuals Using the Mann Whitney U Test. Parameter Microscopy Min Max Mean ± SD 95% CI P-value gSG6-P1 0.0316 Neg 0.00 35730.00 1749.00±2169.00 1503.00-1996.00 Pos 0.00 5697.00 1506.00±907.00 1403.00-1609.00 Pfs230 0.1285 Neg 0.00 6395.00 3275.00±4516.00 2726.00-3788.00 Pos 0.00 34074.00 3203.00±231.60 2747.00-3658.00 EBA 175 3R <0.0001 Neg 0.00 8433172.00 100188.00±673345.00 23684.00-176693.00 Pos 0.00 247994.00 13506.00±28925.00 10214.00-16798.00 Pos; Positive malaria microscopy sample, Neg; Negative malaria microscopy samples, Min; Minimum, Max; Maximum, Mean ± SD; Mean ± Standard Deviation, 95%CI; 95% Confidence Interval, P<0.05 implies statistically significant. Table 5 shows a nationwide comparison of the levels of IgG for gSG6-P1, Pfs230, and EBA 175 3R antigens in malaria microscopy positive and negative samples using the Mann-Whitney U test. 42 University of Ghana http://ugspace.ug.edu.gh The difference between the levels of IgG for Pfs230 was insignificant when the positive and negative samples were compared. Yet it should be noted that averagely, the levels of IgG for Pfs230 in negative malaria microscopy (3275.00±4516.00) samples were higher than in positive malaria microscopy samples (3275.00±4516.00). The levels of IgG for gSG6-P1 and EBA 175 3R were significantly higher in malaria microscopy negative samples (1749.00±2169.00 and 100188.00±673345.00, respectively) compared to average IgG levels for the same antigens in positive malaria microscopy samples (1506.00±907.00 and 13506.00±28925.00 respectively). Objective 2: TO DETERMINE THE DIFFERENT HAEMOGLOBIN GENOTYPES IN MALARIA MICROSCOPY POSITIVE PATIENTS Figure 4: Regional Distribution of Individuals with Haemoglobin Variants Figure 4 shows the haemoglobin variants observed among the malaria microscopy-positive samples from eight regions. In all regions except the Western region, the dominant haemoglobin variant observed from the samples was the HbAA. However, the dominant haemoglobin variant in the Western region was the HbAS. Notwithstanding, HbAC was the second most 43 University of Ghana http://ugspace.ug.edu.gh dominant haemoglobin variant observed across the regions comparatively. The least observed haemoglobin variant observed was the HbCC. Table 6: Kruskal Wallis Variational Test Between the Haemoglobin Variants and the Concentration of IgG’s measured in Microscopy Positive Samples. Parameter HB Min Max Mean ± SD 95% CI P-value Variants gSG6-P1 0.7376 AA 0.00 5696.60 1555.10±987.06 1414.20-1696.00 AC 592.37 2930.90 1667.0±840.75 1159.00-2175.10 AS 506.41 3653.40 1505.40±862.29 1183.40-1827.40 Pfs230 0.6199 AA 0.00 34074.00 2491.60±3232.20 2030.30-2952.90 AC 0.00 3803.50 1718.70±835.39 1213.90-2223.50 AS 0.00 6214.40 2841.70±1681.40 1853.90-3109.60 EBA 175 3R 0.1934 AA 0.00 247994.00 15159.00±34125.00 10276.00-20043.00 AC 0.00 20673.00 5312.00±5429.40 2031.10-8592.90 AS 860.71 111745.00 12133.00±20610.00 4437.10-19829.00 Min; Minimum, Max; Maximum, Mean ± SD; Mean ± Standard Deviation, 95%CI; 95% Confidence Interval, P<0.05 implies statistically significant. Table 6 Compares the haemoglobin variants (HbAA, HbAC, and HbAS) to the IgG levels for the antigens. It was observed that none of the IgG measured for the antigens in the aforementioned haemoglobin variants was significantly higher. That notwithstanding, it could be observed that the IgG levels for all antigens were higher in HbAA samples, followed by HbAS and then HbAC. 44 University of Ghana http://ugspace.ug.edu.gh Objectives 3 and 4: TO DETERMINE THE SEROPREVALENCE OF THREE MALARIA ANTIGENS IN SYMPTOMATIC MALARIA PATIENTS AND THE RELATIONSHIP BETWEEN HAEMOGLOBIN VARIANTS AND IgG LEVELS Figure 5: Seroprevalence of IgG Antibodies for gSG6-P1, Pfs 230, and EBA 175 3R antigens in Microscopy Positive and Negative Samples. Microscopy Negative Microscopy Positive 350 300 250 200 150 100 50 0 Negative Postive Negative Postive Negative Postive gSG6-P1 Pfs230 EBA 175 3R IgG Antibodies in Positive and Negative Microscopy Samples Figure 5 above shows the IgG seroprevalence for the various antigens in microscopy positive and negative samples. The antibody presence was high in both microscopy positive and negative samples regardless of the antigen measured. For example, the positive IgG recorded for EBA 175 3R antigens were 97.7 % and 96.0 % in microscopy-positive and negative samples. Whiles, samples without IgG recorded for the same antigen were 2.3 % and 4.0 % in microscopy positive and negative samples, respectively. 45 Seroprevalence 5 7 295 293 10 10 290 290 7 12 293 288 University of Ghana http://ugspace.ug.edu.gh Table 7: Seroprevalence of IgG Antibodies for gSG6-P1, Pfs230, and EBA 175 3R antigens in Microscopy Positive and Negative Samples Stratified Across Gender, Age, and HB Phenotype. Microscopy Positive Parameter gSG6-P1 P Pfs230 P EBA 175 3R P NEG POS NEG POS NEG POS Gender 0.720 >0.999 0.082 Male 4 (57.1 %) 144 (49.1 %) 5 (50.0 %) 143 (49.3 %) 9 (75.0 %) 139 (48.3 %) Female 3(42.9 %) 149 (50.9 %) 5 (50.0 %) 147 (50.7 %) 3 (25.0 %) 149 (51.7 %) Age 0.478 0.391 0.621 <21 yrs. 6 (85.7 %) 200 (68.3 %) 5 (50.0 %) 201 (69.3 %) 8 (66.7 %) 198 (68.8 %) 21-40 yrs. 0 (0.0 %) 49 (16.7 %) 3 (30.0 %) 46 (15.9 %) 3 (25.0 %) 46 (16.0 %) >40yrs. 1 (14.3 %) 44 (15.0 %) 2 (20.0 %) 43 (14.8 %) 1 (8.3 %) 44 (15.3 %) HB Phenotype 0.001 0.966 0.832 AA 5 (83.3 %) 186 (79.8 %) 6 (75.0 %) 185 (80.1 %) 6 (85.7 %) 185 (79.2 %) AC 0 (0.0 %) 13 (5.6 %) 1 (12.5 %) 12 (5.2 %) 1 (14.3 %) 12 (5.2 %) SC 0 (0.0 %) 2 (0.9 %) 0 (0.0 %) 2 (0.9 %) 0 (0.0 %) 2 (0.9 %) AS 0 (0.0 %) 30 (12.9 %) 1 (12.5 %) 29 (12.6 %) 0 (0.0 %) 30 (12.9 %) CC 0 (0.0 %) 1 (0.4 %) 0 (0.0 %) 1 (0.4 %) 0 (0.0 %) 1 (0.4 %) AF 1 (16.7 %) 1 (0.4 %) 0 (0.0 %) 2 (0.9 %) 0 (0.0 %) 2 (0.9 %) Microscopy Negative Parameter gSG6-P1 P Pfs230 P EBA 175 3R P NEG POS NEG POS NEG POS Gender 0.657 0.751 >0.999 Male 1 (20.0 %) 107 (36.3 %) 4 (40.0 %) 104 (35.9 %) 2 (28.6 %) 106 (36.2 %) Female 4 (80.0 %) 188 (63.7 %) 6 (60.0 %) 186 (64.1 %) 5 (71.4 %) 187 (63.8 %) Age 0.937 0.706 0.471 <21 yrs. 2 (40.0 %) 142 (48.1 %) 6 (60.0 %) 138 (47.6 %) 4 (57.1 %) 140 (47.8 %) 21-40 yrs. 2 (40.0 %) 102 (34.6 %) 3 (30.0 %) 101 (34.8 %) 1 (14.3 %) 103 (35.2 %) >40yrs. 1 (20.0 %) 51 (17.3 %) 1 (10.0 %) 51 (17.6 %) 2 (28.6 %) 50 (17.1 %) P<0.05 implies statistical significance. POS -POSITIVE, NEG -NEGATIVE, P – p-value 49 University of Ghana http://ugspace.ug.edu.gh Table 7 above shows the seroprevalence of the IgG antibodies for gSG6-P1, Pfs230, and EBA 175 3R antigens in microscopy positive and negative samples stratified across gender, age, and HB phenotype. The positive microscopy samples established no significant association among the parameters and the antibodies for the three antigens except for the Hb phenotype in gSG6-P1 antibodies. Thus, HbAA was significantly higher for gSG6-P1 positive antibodies 186(79.8 %) than all other phenotypes in positive microscopy samples. Also, samples without gSG6-P1 antibodies were significantly higher in Hb AA samples 5 (83.3 %) than all other phenotypes. Generally, among the males and females, antibodies for the antigens were higher in the females than in the males in the positive microscopy samples. Although these were statistically insignificant. On the negative microscopy samples, a similar pattern was observed in the presence of antibodies for the antigens among the male and female participants. These were also statistically insignificant. Age-wise, the most seroprevalent antibodies were observed among participants aged <21years for all antigens in the negative microscopy samples. 50 University of Ghana http://ugspace.ug.edu.gh CHAPTER 5 DISCUSSION, CONCLUSION, AND RECOMMENDATION 5.1 DISCUSSION As part of the millennium development goals, malaria, an ancient human pathogen troubling about half of the world's population for eons, needs eradication. This plan has caused an increase in scientific research aimed at developing a vaccine for the disease or possible targeted treatment for specific individuals. This study sought to add to the pool of scientific data available by looking at the effects of haemoglobin variants on the immune response elicited by people with clinically symptomatic malaria and the seroprevalence of these immunoglobulins in the ten regions of Ghana. The most prevalent Hb phenotypes identified were HbAA (79.9 %), HbAS (12.6 %), and HbAC (5.4 %), with none of the other variants identified exceeding 1%. This study of haemoglobin variant distribution agrees with some earlier studies in Ghana but disagrees with the findings from another study in Ghana, where HbAS dominated (Awaitey, 2020). An. gambiae Salivary Gland Protein-6 peptide 1 (gSG6-P1) Recent research has shown that measuring antibody (Ab) responses to vector saliva in human populations could be a valuable biomarker for determining human exposure to vector bites and, consequently, the risk of transmission of vector-borne diseases. (Sadia-Kacou, 2019; Sagna, 2013). Therefore, the seroprevalence, the levels of concentrations, and distribution of An. gambiae Salivary Gland Protein-6 peptide one antibodies (anti-gSG6-P1) determined in this study were not farfetched. Of the samples analyzed, 98 % of the participants produced IgG response to gSG6-P1 salivary peptide. This finding was higher than the 50-60 % of IgG 51 University of Ghana http://ugspace.ug.edu.gh responses to gSG6-P1 in an earlier study conducted in Ghana (Badu, 2015) but similar to the IgG responses to the gSG6-P1 peptide in studies from Burkina Faso and Cameroon (Cheteug, 2020; Faso, 2018). The generally high levels of anti- gSG6-P1 antibodies recorded in this study agree with previous deductions that the level of vector exposure contributes to a decrease or increase in the concentration of the anti- gSG6-P1 antibodies above the detectable threshold. Therefore, people with limited or no exposure to the vector will have very low or no IgG to the gSG6-P1 salivary peptide, and this could be a result of their geographical location, usage of treated insecticide nets, sewage disposal practices, season, and other socio-demographical factors (Cheteug, 2020; Traoré, 2019). This data consequently confirms that the antibody response to the gSG6-P1 peptide is a pertinent marker to assess human exposure to Anopheles mosquitoes. Moreover, the seroprevalence of anti-gSG6-P1 antibodies was insignificantly higher in microscopy, negative 98.3 % and positive individuals 97.7 %. This study was dissimilar to a previous Cameroonian survey where infected individuals had higher levels of antibodies than uninfected individuals (Cheteug, 2020). Further disparity with previous studies linked infection to a rise in anti-gSG6-P1 antibodies (Badu, 2012; Traoré, 2019). Perhaps the presence of the IgG in negative malaria microscopy individuals results from the general persistent exposure to the mosquito vector by participants in this study. Furthermore, an observation of the sero-distribution of gSG6-P1 antibodies among male and female participants in both malaria-positive and negative microscopy individuals from this study was insignificant. This confirms earlier findings that antibody responses to gSG6-P1 peptides are not gender-specific (Cheteug, 2020; Londono-Renteria, 2015; Ya-umphan, 2017). No significant IgG responses to gSG6-P1 peptides were observed within age groups. This 52 University of Ghana http://ugspace.ug.edu.gh study disagrees with an earlier study in Southeast Asia, where aging directly correlated with the increase in IgG responses to the salivary peptide. Thus, further disagreeing with the assertion by the earlier study, which reported that the anti-gSG6-P1 IgG response with age is generally consistent with the gradual acquisition of immunity against Anopheles mosquito saliva (Ya-umphan, 2017). However, this study's finding agrees with the statement that children have higher responses to salivary gSG6-p1 proteins. In contrast, adults had diminished antibody responses, suggesting desensitization of the immune response to the salivary proteins (Badu, 2012) since most of the IgG recorded in this study were from those below the age of 20 years. Additionally, the phenotypic distribution of haemoglobin in microscopy-positive individuals from this study showed a significantly greater number of participants with antibodies against the gSG6-P1 were people with the haemoglobin AA phenotype (79.8 %), followed by haemoglobin AS (12.9 %) and then AC (5.6 %). Plasmodium falciparum surface protein (Pfs 230) A serological assessment of the distribution, prevalence, and concentration levels of immunoglobulins against the Pfs230 antigens in clinically malaria-positive and negative microscopy samples indicated that out of the participants recruited, 96.7 % of them had detectable levels of the antigens. Nevertheless, the levels in malaria-negative microscopy samples (3275.00±4516.00) were more significant than those observed in malaria-positive microscopy samples (3203.00±231.60). A regional comparison of the levels of concentration of IgG against Pfs230 reported most regions had no comparative difference in anti-Pfs230 IgG concentrations between the positive and negative individuals. The seroprevalence of 96.7 % of this sexual stage antibody observed in this study was more significant than the 28.6% reported from Burkina Faso (Ouédraogo, 2011). The high prevalence of anti-Pfs230 antibodies 53 University of Ghana http://ugspace.ug.edu.gh expressed in this study could result from submicroscopic gametocytes in the participants. Thus, the increase in anti-Pfs230 IgG was expected as antibody responses to gametocyte antigens have been suggested to be influenced more by recent exposure (Acquah, 2020; Amoah, 2018), and participants in this study, considering both microscopically reported positive and negatives samples may have had recent gametocytes infection which was not detected by microscopy. Perhaps a molecular approach for detecting submicroscopic gametocytaemia in both the microscopically infected and uninfected participants would have provided information on why most of the participants, regardless of the infection status by microscopy, presented with the antibodies (Bousema, 2006). Although no significant associations were established between the number of participants with detectable levels of Pfs230 antigens in malaria microscopy positive and negative samples across gender, age, or haemoglobin phenotype, it is interesting to note that female participants aged below 21 years and haemoglobin AA phenotype recorded higher numbers of participants with antigen present at a detectable level. The age-antibody response pattern presented in this study disagrees with the findings presented by Stone et al. in 2018, which stipulated those antibodies against the sexual stage-specific antigens positively correlated with age (Stone, 2018). That notwithstanding, the age-sexual stage antibody responses presented in previous studies have been inconclusive as no consensus has been drawn in previous studies on the association of the sexual stage immunity to the presence of infection (Bousema, 2010; Bousema, 2006; Ouédraogo, 2011). Sexual stage antibody responses in participants < 20 years are likely to result from high gametocyte exposure in this age group, especially among children. In participants >21 years, gametocyte exposure may be the lowest. Still, sexual stage commitment during infections may be relatively increased, and antibody responses to sexual 54 University of Ghana http://ugspace.ug.edu.gh stage antigens may become more long-lived, reflecting the maturation of the immune response (Ouédraogo, 2011). This study further compared the concentration of anti-Pfs230 antibodies measured in the participants with different haemoglobin types. The concentration levels for immunoglobulins against Pfs230 were insignificantly higher among the HbAS participants (2841.70±1681.40) than in the other phenotypes measured. The insignificant association between the haemoglobin phenotypes and the sexual stage-specific antibody Pfs230, the finding of this study could be ground-breaking since a possible controlled cohort study between these haemoglobin variants and immunity against the sexual stage antigen could provide insight into the dynamics of host immune responses. Nevertheless, the concentration of anti-Pfs230 being greater in haemoglobin AS than in AA and AC could suggest that AS individuals may have a lower propensity to contribute to communal malaria transmission patterns (Bougouma, 2014) since the influence of haemoglobin variants (AS and AC) have been implicated in the gametocyte density and the chronicity of infection (Gonçalves, 2017). Perhaps the function of the gamete surface antigen (Pfs230) essential in the formation of oocyst is blocked in by the IgG for Pfs230, thus contributing to the reduced infectivity among haemoglobin AS participants controlling subsequent studies could provide more detailed insight (Eksi, 2006). Erythrocyte binding antigen 175 Region 3 (EBA-175 3R) The assessment of the erythrocytic stage antibody against EBA 175 3R antigens to establish its seroprevalence, distribution, and concentration levels among participants taking into account the influence of their haemoglobin variants, revealed that the total number of participants with detectable levels of anti-EBA 175 3R antibodies present was 96.8 %. These high antibody levels reported in this study correlate with the 85.6 % seroprevalence reported 55 University of Ghana http://ugspace.ug.edu.gh from Obom by Amoah et al. in the Greater Accra Region of Ghana but were definitively higher than 58.0 % presented from Asutsuare in the same study (Amoah, 2018). The differences in antibody concentration from the earlier study were attributed to the low prevalence of malaria in Asutsuare compared to Obom. Conceivably, it is not without merit to speculate that high seroprevalence in this study, irrespective of microscopy results, could be due to undetectable parasitic density at the submicroscopic level. However, it is prudent to note that females, participants ages below 21 years, and the haemoglobin AA Phenotype recorded higher anti- EBA 175 3R antibodies. Interestingly, a comparison of the levels of concentration of antibodies for EBA-175 3R to different haemoglobin variants in microscopy positive samples indicated that antibodies against the EBA 175 3R antigens in HbAA samples (15159.00±34125.00) were higher than in HbAC (5312.00±5429.40) and HbAS (12133.00±20610.00). Concerning age, the results presented from this study compared to that of an earlier study in Nigeria were incongruent. The Nigerian study significantly reported a positive correlation between age and the IgG antibodies for EBA 175 3R. A controlled study considering age and immunity development may provide deeper insights into possible vaccine production targeting the erythrocytic stage of the parasite. Perchance, the high endemicity of malaria, the predominance of HbAA, and the season of sample collection contributed to the significantly increased anti-EBA 175 3R antibodies in the Nigerian study. The similarities of this to that of the earlier ones contributed to the commonalities in the findings. Moreover, although studies have reported that anti-EBA 175 IgG confers immunity to malaria infection, a study in Senegal stated that immunity conferred by the IgG in different Hb variants depends on the IgG subclass (Bwire, 2019; Nafady, 2014; Sarr, 2006). 56 University of Ghana http://ugspace.ug.edu.gh Influence of haemoglobinopathies on malaria Finally, the influence of haemoglobinopathies on reducing the morbidity and mortality of malaria has been associated with the ability of these variants of haemoglobin to reduce parasitic invasion into RBCs (Bunyaratvej, 1992; Chotivanich, 2002; Ifediba, 1995; Taylor, 2013), decreasing in intraerythrocytic growth (Fairhurst, 2003; Pasvol, 1978; Taylor, 2013), enhancing sickling of red blood cells at low oxygen tension (Luzzatto, 1970; Roth, 1978), producing micro-RNA that inhibits the enzymatic activities of the parasites (Chen, 2008; LaMonte, 2012; Sangokoya, 2010), and interfering with the intrinsic pathogenic pathway of the parasite by down-regulating the effect P. falciparum erythrocyte membrane protein 1 (PfEMP1) thus impairing rosetting and co-adherence of infected RBCs to the endothelial membranes (Cholera, 2008). However, the involvement of immunoglobulins in thwarting malaria activity has been inconclusive, as some studies have reported higher seroreactivity to specific antigens while others have not (Taylor, 2013). Therefore, the lack of evidence to support the claim that haemoglobinopathies influence the IgG antibody response at different stages of falciparum malaria is unsurprising. 5.2 CONCLUSION At the end of this study, the most dominant haemoglobin phenotype was Hb AA (79.9 %), followed by AS (12.6 %) and AC (5.4 %). Of the antibodies measured, there were significant differences in the total concentration of anti-EBA 175 3R and anti-gSG6-P1 antibodies in malaria negative and positive microscopy samples. Although no association was established between the concentrations of IgGs measured against the various antigens in different haemoglobin variants in malaria microscopy positive samples, it was clear that the number of 57 University of Ghana http://ugspace.ug.edu.gh participants with IgG against gSG6- P1 antigen was significantly greater in Hb AA participants than in the other haemoglobin variants. Yet the same assessment could not be made for the sexual stage antigen (Pfs230) and the erythrocyte binding antigen (EBA 175 3R). In general, no significant relationships were established between the influence of age, gender, and haemoglobin variants on the anti-Pfs230, anti-EBA 175 3R, and anti-gSG6-P1 antibodies 5.3 RECOMMENDATION The study was limited by the lack of a molecular approach to detecting the prevalence of malaria in negative microscopy individuals. Thus, ruling out the possibility of these participants having infections at the sub-microscopic level. Therefore, further study should be conducted considering molecular techniques to detect malaria parasites. Furthermore, a controlled longitudinal study that will consider the vector exposure level, haemoglobin density, and prevention of reinfection among the different haemoglobin variants may untie the gordian knot involved in malaria pathogenicity. Finally, a possible look at the specific subclasses of IgG of the antibodies produced in the different stages of malaria may be the key to creating a vaccine. 58 University of Ghana http://ugspace.ug.edu.gh REFERENCES Abbas, A. K., Murphy, K. M., & Sher, A. (1996). Functional diversity of helper T lymphocytes. Nature, 383(6603), 787–793. Acquah, F.K., Donu, D., Bredu, D. et al. (2020). Asymptomatic carriage of Plasmodium falciparum by individuals with variant blood groups and haemoglobin genotypes in southern Ghana. Malar J 19, 217 Aidoo, M., Terlouw, D. J., Kolczak, M. S., McElroy, P. D., Ter Kuile, F. O., Kariuki, S., Nahlen, B. L., Lal, A. A., & Udhayakumar, V. (2002). Protective effects of the sickle cell gene against malaria morbidity and mortality. Lancet (London, England), 359(9314), 1311–1312. Aikawa, M., Miller, L. H., Johnson, J., & Rabbege, J. (1978). Erythrocyte entry by malarial parasites. A moving junction between erythrocyte and parasite. The Journal of cell biology, 77(1), 72–82. Allen, S. J., O'Donnell, A., Alexander, N. D., Alpers, M. P., Peto, T. E., Clegg, J. B., & Weatherall, D. J. (1997). alpha+-Thalassemia protects children against diseases caused by other infections, as well as malaria. Proceedings of the National Academy of Sciences of the United States of America, 94(26), 14736–14741. Amaratunga, C., Lopera-Mesa, T. M., Brittain, N. J., Cholera, R., Arie, T., Fujioka, H., Keefer, J. R., & Fairhurst, R. M. (2011). A role for fetal haemoglobin and maternal immune IgG in infant resistance to Plasmodium falciparum malaria. PloS one, 6(4), e14798. Amoah, L. E., Abagna, H. B., Akyea-Mensah, K., Lo, A. C., Kusi, K. A., & Gyan, B. A. (2018a). Characterization of anti-EBA175RIII-V in asymptomatic adults and children living in communities in the Greater Accra Region of Ghana with varying 59 University of Ghana http://ugspace.ug.edu.gh malaria transmission intensities. BMC immunology, 19(1), 34. Amoah, L.E., Acquah, F.K., Ayanful-Torgby, R. et al. Dynamics of anti-MSP3 and Pfs230 antibody responses and multiplicity of infection in asymptomatic children from southern Ghana. Parasites Vectors 11, 13 (2018b) Artavanis-Tsakonas, K., & Riley, E. M. (2002). Innate immune response to malaria: rapid induction of IFN-gamma from human NK cells by live Plasmodium falciparum- infected erythrocytes. Journal of immunology (Baltimore, Md.: 1950), 169(6), 2956–2963. Astagneau, P., Roberts, J. M., Steketee, R. W., Wirima, J. J., Lepers, J. P., & Deloron, P. (1995). Antibodies to a Plasmodium falciparum blood-stage antigen as a tool for predicting the protection levels of two malaria-exposed populations. The American Journal of tropical medicine and hygiene, 53(1), 23–28. Awaitey, D. K., Akorsu, E. E., Allotey, E. A., Kwasie, D. A., Kwadzokpui, P. K., Tawiah, P. A., Amankwah, S. A., & Abaka-Yawson, A. (2020). Assessment of Haemoglobin Variants in Patients Receiving Health Care at the Ho Teaching Hospital: A Three-Year Retrospective Study. Advances in haematology, 2020, 7369731. Badu, K., Gyan, B., Appawu, M., Mensah, D., Dodoo, D., Yan, G., Drakeley, C., Zhou, G., Owusu-Dabo, E., & Koram, K. A. (2015). Serological evidence of vector and parasite exposure in Southern Ghana: the dynamics of malaria transmission intensity. Parasites & vectors, 8, 251. Baidjoe, A., Stone, W., Ploemen, I. et al. Combined DNA extraction and antibody elution from filter papers for the assessment of malaria transmission intensity in epidemiological studies. Malar J 12, 272 (2013). 60 University of Ghana http://ugspace.ug.edu.gh Ballet, J. J., Druilhe, P., Vasconcelos, I., Schmitt, C., Agrapart, M., & Frommel, D. (1985). Human lymphocyte responses to Plasmodium falciparum merozoite antigens. A functional assay of protective immunity. Transactions of the Royal Society of Tropical Medicine and Hygiene, 79(4), 497–499. Bardakdjian-Michau, J., Dhondt, J. L., Ducrocq, R., Galactéros, F., Guyard, A., Huchet, F. X., Lahary, A., Lena-Russo, D., Maboudou, P., North, M. L., Prehu, C., Soummer, A. M., Verschelde, M., Wajcman, H., & Groupe de travail SFBC << Recommandations dans le domaine des diagnostics des hémoglobinopathies >> (2003). Bonnes pratiques de l'étude de l'hémoglobine [Good practices for the study of haemoglobin]. Annales de biologie clinique, 61(4), 401–409. Bartoloni, A., & Zammarchi, L. (2012). Clinical aspects of uncomplicated and severe malaria. Mediterranean Journal of haematology and infectious diseases, 4(1), e2012026. https://doi.org/10.4084/MJHID.2012.026 Baruch DI, Gormely JA, Ma C, Howard RJ, Pasloske BL (1996) Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1. Proc Natl Acad Sci U S A 93: 3497–3502. Bejon, P., Lusingu, J., Olotu, A., Leach, A., Lievens, M., Vekemans, J., Mshamu, S., Lang, T., Gould, J., Dubois, M. C., Demoitié, M. A., Stallaert, J. F., Vansadia, P., Carter, T., Njuguna, P., Awuondo, K. O., Malabeja, A., Abdul, O., Gesase, S., Mturi, N., … von Seidlein, L. (2008). Efficacy of RTS,S/AS01E vaccine against malaria in children 5 to 17 months of age. The New England journal of medicine, 359(24), 2521–2532. Bertram H. Lubin, H. Ewa Witkowska, Klara Kleman, Laboratory diagnosis of 61 University of Ghana http://ugspace.ug.edu.gh haemoglobinopathies, Clinical Biochemistry, Volume 24, Issue 4 1991, Pages 363- 374, Bissé, E., Schaeffer-Reiss, C., Van Dorsselaer, A., Alayi, T. D., Epting, T., Winkler, K., Benitez Cardenas, A. S., Soman, J., Birukou, I., Samuel, P. P., & Olson, J. S. (2017). Haemoglobin Kirklareli (α H58L), a New Variant Associated with Iron Deficiency and Increased CO Binding. The Journal of biological chemistry, 292(6), 2542–2555. Blackman, M. J., Scott-Finnigan, T. J., Shai, S., & Holder, A. A. (1994). Antibodies inhibit the protease-mediated processing of a malaria merozoite surface protein. The Journal of experimental medicine, 180(1), 389–393. Bougouma, E.C., Tiono, A.B., Ouédraogo, A. et al.(2012) Haemoglobin variants and Plasmodium falciparum malaria in children under five years of age living in a high and seasonal malaria transmission area of Burkina Faso. Malar J 11, 154. Bouharoun-Tayoun, H., Attanath, P., Sabchareon, A., Chongsuphajaisiddhi, T., & Druilhe, P. (1990). Antibodies that protect humans against Plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro but act in cooperation with monocytes. The Journal of experimental medicine, 172(6), 1633– 1641. Bousema, J. T., Roeffen, W., van der Kolk, M., de Vlas, S. J., van de Vegte-Bolmer, M., Bangs, M. J., Teelen, K., Kurniawan, L., Maguire, J. D., Baird, J. K., & Sauerwein, R. W. (2006). Rapid onset of transmission-reducing antibodies in Javanese migrants exposed to malaria in Papua, Indonesia. The American Journal of tropical medicine and hygiene, 74(3), 425–431. Bousema, T., Roeffen, W., Meijerink, H., Mwerinde, H., Mwakalinga, S., van Gemert, G. 62 University of Ghana http://ugspace.ug.edu.gh J., van de Vegte-Bolmer, M., Mosha, F., Targett, G., Riley, E. M., Sauerwein, R., & Drakeley, C. (2010). The dynamics of naturally acquired immune responses to Plasmodium falciparum sexual stage antigens Pfs230 & Pfs48/45 in a low endemic area in Tanzania. PloS one, 5(11), e14114. Brockelman CR, Wongsattayanont B, Tan-ariya P, Fucharoen S (1987) Thalassemic erythrocytes inhibit in vitro growth of Plasmodium falciparum. J Clin Microbiol 25: 56–60 Brown, J., Greenwood, B. M., & Terry, R. J. (1986). Cellular mechanisms involved in recovery from acute malaria in Gambian children. Parasite immunology, 8(6), 551– 564. Bull, P. C., Lowe, B. S., Kortok, M., Molyneux, C. S., Newbold, C. I., & Marsh, K. (1998). Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria. Nature medicine, 4(3), 358–360. Bull, P., & ABDI, A. (2016). The role of PfEMP1 as targets of naturally acquired immunity to childhood malaria: Prospects for a vaccine. Parasitology, 143(2), 171-186. Bunyaratvej A, Butthep P, Fucharoen S, Saw D:(1992a) Erythrocyte Volume and Haemoglobin Concentration in Haemoglobin H Disease: Discrimination between the Two Genotypes. Acta Haematol 1992;87:1-5. Bunyaratvej A, Butthep P, Sae-Ung N, Fucharoen S, Yuthavong Y (1992b) Reduced deformability of thalassemic erythrocytes and erythrocytes with abnormal haemoglobins and relation with susceptibility to Plasmodium falciparum invasion. Blood 79: 2460–2463. Bwire, G.M., Majigo, M., Makalla, R. et al.(2019) Immunoglobulin G responses against falciparum malaria-specific antigens are higher in children with homozygous sickle 63 University of Ghana http://ugspace.ug.edu.gh cell trait than those with normal heamoglobin. BMC Immunol 20, 12 Camus, D. & Hadley, T. J. (1985). A Plasmodium falciparum antigen binds to host erythrocytes and merozoites. Science 230, 553–556. Carlson J, Helmby H, Hill AV, Brewster D, Greenwood BM, et al. (1990) Human cerebral malaria: association with erythrocyte rosetting and lack of anti-rosetting antibodies. Lancet 336: 1457–1460. Celada A, Cruchaud A, Perrin LH (1982) Opsonic activity of human immune serum on in vitro phagocytosis of Plasmodium falciparum-infected red blood cells by monocytes. Clin Exp Immunol 47: 635–644. Centers for Disease Control and Prevention. (2020) Malaria Worldwide. http://www.cdc.gov/malaria/malariaworldwide/index.html. Chen Q, Barragan A, Fernandez V, Sundstrom A, Schlichtherle M, et al. (1998) Identification of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) as the rosetting ligand of the malaria parasite P. falciparum. J Exp Med 187: 15–23. Chen, E., Paing, M. M., Salinas, N., Sim, B. K., & Tolia, N. H. (2013). Structural and functional basis for inhibition of erythrocyte invasion by antibodies that target Plasmodium falciparum EBA-175. PLoS pathogens, 9(5), e1003390. Chen, S. Y., Wang, Y., Telen, M. J., & Chi, J. T. (2008). The genomic analysis of erythrocyte microRNA expression in sickle cell diseases. PloS one, 3(6), e2360. Cheteug, G., Elanga-Ndille, E., Donkeu, C., Ekoko, W., Oloume, M., Essangui, E., Nwane, P., NSango, S. E., Etang, J., Wanji, S., Ayong, L., & Eboumbou Moukoko, C. E. (2020). Preliminary validation of the use of IgG antibody response to Anopheles gSG6-p1 salivary peptide to assess human exposure to malaria vector bites in two 64 University of Ghana http://ugspace.ug.edu.gh endemic areas of Cameroon in Central Africa. PloS one, 15(12), e0242510. Cholera R, Brittain NJ, Gillrie MR, Lopera-Mesa TM, Diakite SA, et al. (2008) Impaired cytoadherence of Plasmodium falciparum-infected erythrocytes containing sickle haemoglobin. Proc Natl Acad Sci U S A 105: 991–996. Chotivanich K, Udomsangpetch R, Pattanapanyasat K, Chierakul W, Simpson J, et al. (2002) Haemoglobin E: a balanced polymorphism protective against high parasitemias and thus severe P falciparum malaria. Blood 100: 1172–1176. Clark IA, Awburn MM, Harper CG, Liomba NG, Molyneux ME (2003) Induction of HO- 1 in tissue macrophages and monocytes in fatal falciparum malaria and sepsis. Malar J 2: 41. Cohen S, Mc GI, Carrington S (1961) Gamma-globulin and acquired immunity to human malaria. Nature 192: 733–737. Cox-Singh, J., Davis, T. M., Lee, K. S., Shamsul, S. S., Matusop, A., Ratnam, S., Rahman, H. A., Conway, D. J., & Singh, B. (2008). Plasmodium knowlesi malaria in humans is widely distributed and potentially life-threatening. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America, 46(2), 165– 171. Creary, M., Williamson, D., & Kulkarni, R. (2007). Sickle cell disease: current activities, public health implications, and future directions. Journal of women's health (2002), 16(5), 575–582. Crompton, P.D., J Clin Invest Pierce, S.K., Miller, L.H. (2010). Advances and challenges in malaria vaccine development. 120: 4168–4178. Cyrklaff M, Sanchez CP, Kilian N, Bisseye C, Simpore J, et al. (2011) Haemoglobins S and C interfere with actin remodeling in Plasmodium falciparum-infected 65 University of Ghana http://ugspace.ug.edu.gh erythrocytes. Science 334: 1283–1286. Danquah, I., Ziniel, P., Eggelte, T. A., Ehrhardt, S., & Mockenhaupt, F. P. (2010). Influence of haemoglobins S and C on predominantly asymptomatic Plasmodium infections in northern Ghana. Transactions of the Royal Society of Tropical Medicine and Hygiene, 104(11), 713–719. De Caterina R, Libby P, Peng HB, Thannickal VJ, Rajavashisth TB, et al. (1995) Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J Clin Invest 96: 60–68. Drakeley, C., Sutherland, C., Bousema, J. T., Sauerwein, R. W., & Targett, G. A. (2006). The epidemiology of Plasmodium falciparum gametocytes: weapons of mass dispersion. Trends in parasitology, 22(9), 424–430. https://doi.org/10.1016/j.pt.2006.07.001 Druilhe, P., & Pérignon, J. L. (1994). Mechanisms of defense against P. falciparum asexual blood stages in humans. Immunology letters, 41(2-3), 115–120. Druilhe, P., Pradier, O., Marc, J.P., Miltgen, F., Mazier, D., and Parent, G., (1986). Levels of antibodies to Plasmodium falciparum sporozoite surface antigens reflect malaria transmission rates and are persistent in the absence of reinfection. Infect. Immun.53, 393–397 Duraisingh, M. T., Maier, A. G., Triglia, T., & Cowman, A. F. (2003a). Erythrocyte- binding antigen 175 mediates invasion in Plasmodium falciparum utilizing sialic acid-dependent and -independent pathways. Proceedings of the National Academy of Sciences of the United States of America, 100(8), 4796–4801. Duraisingh, M. T., Triglia, T., Ralph, S. A., Rayner, J. C., Barnwell, J. W., McFadden, G. 66 University of Ghana http://ugspace.ug.edu.gh I., & Cowman, A. F. (2003b). Phenotypic variation of Plasmodium falciparum merozoite proteins directs receptor targeting for invasion of human erythrocytes. The EMBO journal, 22(5), 1047–1057. Eksi, S., Czesny, B., van Gemert, G. J., Sauerwein, R. W., Eling, W., & Williamson, K. C. (2006). Malaria transmission-blocking antigen, Pfs230, mediates human red blood cell binding to exflagellating male parasites and oocyst production. Molecular microbiology, 61(4), 991–998. Eksi, S., Stump, A., Fanning, S.L., Shenouda, M.I., Fujioka, H., and Williamson, K.C. (2002) Targeting and sequestration of truncated Pfs230 in an intraerythrocytic compartment during Plasmodium falciparum gametocytogenesis.Mol Microbiol 44: 1507–1516. Erbayraktar Z, Erbayraktar S, Yilmaz O, Cerami A, Coleman T, et al. (2009) Nonerythropoietic tissue protective compounds are highly effective facilitators of wound healing. Mol Med 15: 235–241 Esposito, F., Lombardi, S., Modiano, D., Zavala, F., Reeme, J., Lamizana, L., Coluzzi, M., Nussenzweig, R.S., (1988). Prevalence and levels of antibodies to the circumsporozoite protein of Plasmodium falciparum in an endemic area and their relationship to resistance to malaria infection. Trans. R. Soc. Trop. Med. Hyg.82, 827–832 Fairhurst RM, Baruch DI, Brittain NJ, Ostera GR, Wallach JS, et al. (2005) Abnormal display of PfEMP-1 on erythrocytes carrying haemoglobin C may protect against malaria. Nature 435: 1117–1121. Fairhurst RM, Fujioka H, Hayton K, Collins KF, Wellems TE (2003) Aberrant development of Plasmodium falciparum in haemoglobin CC red cells: implications 67 University of Ghana http://ugspace.ug.edu.gh for the malaria protective effect of the homozygous state. Blood 101: 3309–3315. Farrance, C. E., Rhee, A., Jones, R. M., Musiychuk, K., Shamloul, M., Sharma, S., Mett, V., Chichester, J. A., Streatfield, S. J., Roeffen, W., van de Vegte-Bolmer, M., Sauerwein, R. W., Tsuboi, T., Muratova, O. V., Wu, Y., & Yusibov, V. (2011). A plant-produced Pfs230 vaccine candidate blocks the transmission of Plasmodium falciparum. Clinical and vaccine immunology: CVI, 18(8), 1351–1357. Felger, I., Maire, M., Bretscher, M. T., Falk, N., Tiaden, A., Sama, W., Beck, H. P., Owusu- Agyei, S., & Smith, T. A. (2012). The dynamics of natural Plasmodium falciparum infections. PloS one, 7(9), e45542. Fell, A. H., & Smith, N. C. (1998). Immunity to asexual blood stages of Plasmodium: is resistance to acute malaria adaptive or innate. Parasitology today (Personal ed.), 14(9), 364–369. Friedman MJ, Roth EF, Nagel RL, Trager W (1979) The role of haemoglobins C, S, and Nbalt in the inhibition of malaria parasite development in vitro. Am J Trop Med Hyg 28: 777–780 Galacteros, F. (1986) Thalassémie, drépanocytose et autres hémoglobinopathies. Techniques et Biologie, 3, 174-178 Garrison, Ingrid (2015). Reportable Infectious Diseases in Kansas 2012 Summary. Kansas: Bureau of Epidemiology and Public Health Informatics. Giardine, B., Borg, J., Viennas, E., Pavlidis, C., Moradkhani, K., Joly, P., Bartsakoulia, M., Riemer, C., Miller, W., Tzimas, G., Wajcman, H., Hardison, R. C., & Patrinos, G. P. (2014). Updates of the HbVar database of human haemoglobin variants and thalassemia mutations. Nucleic acids research, 42(Database issue), D1063–D1069. Good, M. F., & Doolan, D. L. (1999). Immune effector mechanisms in malaria. Current 68 University of Ghana http://ugspace.ug.edu.gh opinion in immunology, 11(4), 412–419. Groux, H., & Gysin, J. (1990). Opsonization as an effector mechanism in human protection against asexual blood stages of Plasmodium falciparum: functional role of IgG subclasses. Research in immunology, 141(6), 529–542. Hall A. P. (1977). The treatment of severe falciparum malaria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 71(5), 367–378. Haynes, J. D., Diggs, C. L., Hines, F. A., & Desjardins, R. E. (1976). Culture of human malaria parasites Plasmodium falciparum. Nature, 263(5580), 767–769. Healer, J., McGuinness, D., Carter, R., & Riley, E. (1999). Transmission-blocking immunity to Plasmodium falciparum in malaria-immune individuals is associated with antibodies to the gamete surface protein Pfs230. Parasitology, 119 ( Pt 5), 425– 433. Hempe, JM Granger, JN and Craver, RD. (1997) Capillary isoelectric focusing of haemoglobin variants. Electrophoresis, 18, 1785-1795 Hirunpetcharat, C., Stanisic, D., Liu, X. Q., Vadolas, J., Strugnell, R. A., Lee, R., Miller, L. H., Kaslow, D. C., & Good, M. F. (1998). Intranasal immunization with yeast- expressed 19 kD carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (yMSP119) induces protective immunity to blood-stage malaria infection in mice. Parasite immunology, 20(9), 413–420. Ho, M., Sexton, M. M., Tongtawe, P., Looareesuwan, S., Suntharasamai, P., & Webster, H. K. (1995). Interleukin-10 inhibits tumor necrosis factor production but not antigen-specific lymphoproliferation in acute Plasmodium falciparum malaria. The Journal of infectious diseases, 172(3), 838–844. Hoffman S. L. (1992). Diagnosis, treatment, and prevention of malaria. The Medical clinics 69 University of Ghana http://ugspace.ug.edu.gh of North America, 76(6), 1327–1355. https://doi.org/10.1016/s0025- 7125(16)30290-5 Holder, A. A., Blackman, M. J., Burghaus, P. A., Chappel, J. A., Ling, I. T., McCallum- Deighton, N., & Shai, S. (1992). A malaria merozoite surface protein (MSP1)- structure, processing, and function. Memorias do Instituto Oswaldo Cruz, 87 Suppl 3, 37–42. Huisman, T.H.J. and Jonxis J.H.P.(1977) The haemoglobinopathies: identification techniques. Marcel Dekker, New York Hunt NH, Grau GE (2003) Cytokines: accelerators and brakes in the pathogenesis of cerebral malaria. Trends Immunol 24: 491–499. Ifediba, T. C., Stern, A., Ibrahim, A., & Rieder, R. F. (1985). Plasmodium falciparum in vitro: diminished growth in haemoglobin H disease erythrocytes. Blood, 65(2), 452–455. Jaramillo M, Plante I, Ouellet N, Vandal K, Tessier PA, et al. (2004) Hemozoin-inducible proinflammatory events in vivo: potential role in malaria infection. J Immunol 172: 3101–3110. Kanter, J., & Kruse-Jarres, R. (2013). Management of sickle cell disease from childhood through adulthood. Blood reviews, 27(6), 279–287. Kapturczak MH, Wasserfall C, Brusko T, Campbell-Thompson M, Ellis TM, et al. (2004) Haem oxygenase-1 modulates early inflammatory responses: evidence from the haem oxygenase-1-deficient mouse. Am J Pathol 165: 1045–1053. Kaul DK, Roth EF Jr, Nagel RL, Howard RJ, Handunnetti SM (1991) Rosetting of Plasmodium falciparum-infected red blood cells with uninfected red blood cells enhances microvascular obstruction under flow conditions. Blood 78: 812–819. 70 University of Ghana http://ugspace.ug.edu.gh Kim H, Higgins S, Liles WC, Kain KC (2011) Endothelial activation and dysregulation in malaria: a potential target for novel therapeutics. Curr Opin Hematol 18: 177–185. Kobayashi, F., Morii, T., Matsui, T., Fujino, T., Watanabe, Y., Weidanz, W. P., & Tsuji, M. (1996). Production of interleukin ten during malaria is caused by lethal and nonlethal variants of Plasmodium yoelii yoelii. Parasitology Research, 82(5), 385– 391. Koh SH, Noh MY, Cho GW, Kim KS, Kim SH (2009) Erythropoietin increases the motility of human bone marrow-multipotent stromal cells (hBM-MSCs) and enhances the production of neurotrophic factors from hBM-MSCs. Stem Cells Dev 18: 411–421. Konaté, L., Zwetyenga, J., Rogier, C., Bischoff, E., Fontenille, D., Tall, A., Spiegel, A., Trape, J. F., & Mercereau-Puijalon, O. (1999). Variation of Plasmodium falciparum msp1 block two and msp2 allele prevalence and infection complexity in two neighboring Senegalese villages with different transmission conditions. Transactions of the Royal Society of Tropical Medicine and Hygiene, 93 Suppl 1, 21–28. Krause MA, Diakite SA, Lopera-Mesa TM, Amaratunga C, Arie T, et al. (2012) alpha- Thalassemia impairs the cytoadherence of Plasmodium falciparum-infected erythrocytes. PLoS One 7: e37214 Krauss, J.S. Drew, P.A. Jonah, M.H. Trinh, M. Shell, S. Black, L.and Baisden, C.R. (1986) Densitometry and micro chromatography compared for determination of the haemoglobin C and A2 proportions in haemoglobin C and haemoglobin SC disease and in haemoglobin C trait. Clin. Chem. 32, 5, 860-863. Krishnegowda G, Hajjar AM, Zhu J, Douglass EJ, Uematsu S, et al. (2005) Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols 71 University of Ghana http://ugspace.ug.edu.gh of Plasmodium falciparum: cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity. J Biol Chem 280: 8606–8616. Kumaratilake, L. M., Ferrante, A., Jaeger, T., & Rzepczyk, C. M. (1992). Effects of cytokines, complement, and antibody on the neutrophil respiratory burst and phagocytic response to Plasmodium falciparum merozoites. Infection and immunity, 60(9), 3731–3738. Kweku, M, Dinko, B et al. (2017).Comparative laboratory diagnosis of malaria using light microscopy and rapid diagnostic test kit in the Hohoe Municipality, Ghana. Journal of Scientific Research and Studies Vol. 4(4), pp. 86-92. Kwiatkowski, D., Cannon, J. G., Manogue, K. R., Cerami, A., Dinarello, C. A., & Greenwood, B. M. (1989). Tumour necrosis factor production in Falciparum malaria and its association with schizont rupture. Clinical and experimental immunology, 77(3), 361–366. LaMonte, G., Philip, N., Reardon, J., Lacsina, J. R., Majoros, W., Chapman, L., Thornburg, C. D., Telen, M. J., Ohler, U., Nicchitta, C. V., Haystead, T., & Chi, J. T. (2012). Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance. Cell host & microbe, 12(2), 187–199. Landers J. P. (1995). Clinical capillary electrophoresis. Clinical chemistry, 41(4), 495–509. Livingstone FB (1967) Abnormal haemoglobins in human populations. Aldine Publishing, Chicago Londono-Renteria, B., Drame, P.M., Weitzel, T. et al. An. gambiae gSG6-P1 evaluation as a proxy for human-vector contact in the Americas: a pilot study. Parasites Vectors 72 University of Ghana http://ugspace.ug.edu.gh 8, 533 (2015). Lopera-Mesa TM, Mita-Mendoza NK, van de Hoef DL, Doumbia S, Konate D, et al. (2012) Plasma Uric Acid Levels Correlate with Inflammation and Disease Severity in Malian Children with Plasmodium falciparum Malaria. PLoS One 7: e46424 Lu, L., Barbi, J., & Pan, F. (2017). The regulation of immune tolerance by FOXP3. Nature reviews. Immunology, 17(11), 703–717. Luchavez, J., Espino, F., Curameng, P., Espina, R., Bell, D., Chiodini, P., Nolder, D., Sutherland, C., Lee, K. S., & Singh, B. (2008). Human Infections with Plasmodium knowlesi, the Philippines. Emerging infectious diseases, 14(5), 811–813. Luzzatto, L., Nwachuku-Jarrett, E. S., & Reddy, S. (1970). Increased sickling of parasitized erythrocytes as a mechanism of resistance against malaria in the sickle-cell trait. Lancet (London, England), 1(7642), 319–321. Luzzi GA, Merry AH, Newbold CI, Marsh K, Pasvol G, et al. (1991) Surface antigen expression on Plasmodium falciparum-infected erythrocytes is modified in alpha- and beta-thalassemia. J Exp Med 173: 785–791. Maier-Redelsberger, M. Girot, R. (1989)Diagnostic biologique des maladies de l’hémoglobine. Feuillets de biologie, 170. McCarra, M. B., Ayodo, G., Sumba, P. O., Kazura, J. W., Moormann, A. M., et al. (2011) Antibodies to Plasmodium falciparum erythrocyte-binding antigen-175 are associated with protection from clinical malaria. Pediatr Infect Dis J 30:1037–1042. MCGREGOR I. A. (1964). THE PASSIVE TRANSFER OF HUMAN MALARIAL IMMUNITY. The American Journal of tropical medicine and hygiene, 13, 237– 239. Ming, W., Qi-Jie, D., Rong-Zhen, L., Ping, T., Chuan, L., & Zhi-Ning, D. (1987). 73 University of Ghana http://ugspace.ug.edu.gh Diagnosis of tertian malaria by enzyme-linked immunosorbent assay. Trans. R. Soc. Trop. Med. Hyg, 81(6), 888–890. Moody A. (2002). Rapid diagnostic tests for malaria parasites. Clinical microbiology reviews, 15(1), 66–78. https://doi.org/10.1128/CMR.15.1.66-78.2002 Nafady, H., Eida, A. and Eida, O. (2014) Immunological Characterization in Malaria Patients with and without the Sickle-Cell Trait. Advances in Infectious Diseases, 4, 152-164. Narum, D. L., Ogun, S. A., Thomas, A. W., & Holder, A. A. (2000). Immunization with parasite-derived apical membrane antigen one or passive immunization with a specific monoclonal antibody protects BALB/c mice against lethal Plasmodium yoelii yoelii YM blood-stage infection. Infection and immunity, 68(5), 2899–2906. Nonvignon, J., Aryeetey, G.C., Malm, K.L., et al. Economic burden of malaria on businesses in Ghana: a case for private sector investment in malaria control. Malar J 15, 454 (2016). Ockenhouse, C. F., Schulman, S., & Shear, H. L. (1984). Induction of crisis forms in the human malaria parasite Plasmodium falciparum by gamma-interferon-activated, monocyte-derived macrophages. Journal of immunology (Baltimore, Md.: 1950), 133(3), 1601–1608. Oda RP et al. Capillary electrophoresis as a clinical tool for the analysis of protein in serum and other body fluids. Electrophoresis, 18, 1715-1723 (1997). Oeuvray, C., Bouharoun-Tayoun, H., Gras-Masse, H., Bottius, E., Kaidoh, T., Aikawa, M., Filgueira, M. C., Tartar, A., & Druilhe, P. (1994). Merozoite surface protein-3 is a malaria protein-inducing antibody that promotes Plasmodium falciparum killing by cooperation with blood monocytes. Blood, 84(5), 1594–1602. 74 University of Ghana http://ugspace.ug.edu.gh Okenu, D.M., Riley, E.M., Bickle, Q.D., Agomo, P.U., …Barbosa, A. (2000). Analysis of human antibodies to erythrocyte binding antigen 175 of Plasmodium falciparumInfect Immun 68: 5559-5566. Olson JA, Nagel RL (1986) Synchronized cultures of P falciparum in abnormal red cells: the mechanism of the inhibition of growth in HbCC cells. Blood 67: 997–1001. Olusegun M. Akanbi, Akhere A. Omonkhua & Mojisola C. Cyril-Olutayo (2014) Effect of Methanolic Extract of Stem Bark of Anogeissus Leiocarpus on Liver Function of Mice Infected with Plasmodium Berghei, Journal of Herbs, Spices & Medicinal Plants, 20:4, 350-358, O'Meara, W. P., Bejon, P., Mwangi, T. W., Okiro, E. A., Peshu, N., Snow, R. W., Newton, C. R., & Marsh, K. (2008). Effect of a fall in malaria transmission on morbidity and mortality in Kilifi, Kenya. Lancet (London, England), 372(9649), 1555–1562. Orago, A. S., & Facer, C. A. (1991). Cytotoxicity of natural human killer (NK) cell subsets for Plasmodium falciparum erythrocytic schizonts: stimulation by cytokines and inhibition by neomycin. Clinical and experimental immunology, 86(1), 22–29. Orkin, S. H., & Bauer, D. E. (2019). Emerging Genetic Therapy for Sickle Cell Disease. Annual review of medicine, 70, 257–271. Osier, F. H. A., G., Polley, S. D., Murungi, L., Verra, F., Tetteh,K. K. A., Lowe, B., …Mwangi, T. (2008). Breadth and magnitude of antibody responses to multiple Plasmodium falciparum merozoite antigens are associated with protection from clinical malaria. Infect Immun 76: 2240–2248. Ouédraogo, A. L., Roeffen, W., Luty, A. J., de Vlas, S. J., Nebie, I., Ilboudo-Sanogo, E., Cuzin-Ouattara, N., Teleen, K., Tiono, A. B., Sirima, S. B., Verhave, J. P., Bousema, T., & Sauerwein, R. (2011). Naturally acquired immune responses to 75 University of Ghana http://ugspace.ug.edu.gh Plasmodium falciparum sexual stage antigens Pfs48/45 and Pfs230 in an area of seasonal transmission. Infection and immunity, 79(12), 4957–4964. Parroche P, Lauw FN, Goutagny N, Latz E, Monks BG, et al. (2007) Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9. Proc Natl Acad Sci U S A 104: 1919–1924. Pasvol G (1980) The interaction between sickle haemoglobin and the malarial parasite Plasmodium falciparum. Trans R Soc Trop Med Hyg 74: 701–705. Pasvol G, Weatherall DJ, Wilson RJ (1978) Cellular mechanism for the protective effect of haemoglobin S against P. falciparum malaria. Nature 274: 701–703. Piper, K. P., Hayward, R. E., Cox, M. J., & Day, K. P. (1999). Malaria transmission and naturally acquired immunity to PfEMP-1. Infection and immunity, 67(12), 6369– 6374. Pollard, E.J.M., Patterson, C., Russell, T.L. et al. Human exposure to Anopheles farauti bites in the Solomon Islands is not associated with IgG antibody response to the gSG6 salivary protein of Anopheles gambiae. Malar J 18, 334 (2019). Ranson, H., & Lissenden, N. (2016). Insecticide Resistance in African Anopheles Mosquitoes: A Worsening Situation that Needs Urgent Action to Maintain Malaria Control. Trends in parasitology, 32(3), 187–196. Rees, D. C., Williams, T. N., & Gladwin, M. T. (2010). Sickle-cell disease. Lancet (London, England), 376(9757), 2018–2031. Richards, J. S., Stanisic, D. I., Fowkes, F. J., Tavul, L., Dabod, E, et al. (2010).Association between naturally acquired antibodies to erythrocyte-binding antigens of Plasmodium falciparum and protection from malaria and high-density parasitemia.Clin Infect Dis 51: e50–60. 76 University of Ghana http://ugspace.ug.edu.gh Robert J. Wilkinson, Geoffrey Pasvol, Host resistance to malaria runs into swampy water, Roth, E. F., Jr, Friedman, M., Ueda, Y., Tellez, I., Trager, W., & Nagel, R. L. (1978). Sickling rates of human AS red cells infected in vitro with Plasmodium falciparum malaria. Science (New York, N.Y.), 202(4368), 650–652. Sabchareon, A., Burnouf, T., Ouattara, D., Attanath, P., Bouharoun-Tayoun, H., Chantavanich, P., Foucault, C., Chongsuphajaisiddhi, T., & Druilhe, P. (1991). Parasitologic and clinical human response to immunoglobulin administration in falciparum malaria. The American journal of tropical medicine and hygiene, 45(3), 297–308. Sachs, J., & Malaney, P. (2002). The economic and social burden of malaria. Nature, 415(6872), 680–685. Sadia-Kacou, C., Yobo, C. M., Adja, M. A., Sagna, A. B., Ndille, E. E., Poinsignon, A., Tano, Y., Koudou, B. G., & Remoue, F. (2019). Use of Anopheles salivary biomarker to assess seasonal variation of human exposure to Anopheles bites in children living near rubber and oil palm cultivations in Côte d'Ivoire. Parasite epidemiology and control, 5, e00102. Sagna, A.B., Sarr, J.B., Gaayeb, L. et al. gSG6-P1 salivary biomarker discriminate micro- geographical heterogeneity of human exposure to Anopheles bites in low and seasonal malaria areas. Parasites Vectors 6, 68 (2013). Sangokoya, C., Telen, M. J., & Chi, J. T. (2010). microRNA miR-144 modulates oxidative stress tolerance and associated with anemia severity in sickle cell disease. Blood, 116(20), 4338–4348. https://doi.org/10.1182/blood-2009-04-214817 Sarr, J. B., Pelleau, S., Toly, C., Guitard, J., Konaté, L., Deloron, P., Garcia, A., & Migot- Nabias, F. (2006). Impact of red blood cell polymorphisms on the antibody 77 University of Ghana http://ugspace.ug.edu.gh response to Plasmodium falciparum in Senegal. Microbes and infection, 8(5), 1260–1268. Schaefer, B. A., Flanagan, J. M., Alvarez, O. A., Nelson, S. C., Aygun, B., Nottage, K. A., George, A., Roberts, C. W., Piccone, C. M., Howard, T. A., Davis, B. R., & Ware, R. E. (2016). Genetic Modifiers of White Blood Cell Count, Albuminuria and Glomerular Filtration Rate in Children with Sickle Cell Anemia. PloS one, 11(10), e0164364. Schellenberg, J. R., Smith, T., Alonso, P. L., & Hayes, R. J. (1994). What is clinical malaria? Finding case definitions for field research in highly endemic areas. Parasitology today (Personal ed.), 10(11), 439–442. https://doi.org/10.1016/0169- 4758(94)90179-1 Schneider R. G. (1978). Methods for detection of hemoglobin variants and hemoglobinopathies in the routine clinical laboratory. CRC critical reviews in clinical laboratory sciences, 9(3), 243–271. Schofield L, Grau GE (2005) Immunological processes in malaria pathogenesis. Nat Rev Immunol 5: 722–735. Schofield L, Hackett F (1993) Signal transduction in host cells by a glycosylphosphatidylinositol toxin of malaria parasites. J Exp Med 177: 145–153. Shear, H. L., Ng, C., & Zhao, Y. (1990). Cytokine production in lethal and non-lethal murine malaria. Immunology letters, 25(1-3), 123–127. Shretta, R., Silal, S.P., Malm, K., et al. Estimating the risk of declining funding for malaria in Ghana: the case for continued investment in the malaria response. Malar J 19, 196 (2020). Siren AL, Fratelli M, Brines M, Goemans C, Casagrande S, et al. (2001) Erythropoietin 78 University of Ghana http://ugspace.ug.edu.gh prevents neuronal apoptosis after cerebral ischemia and metabolic stress. Proc Natl Acad Sci U S A 98: 4044–4049. Smith JD, Chitnis CE, Craig AG, Roberts DJ, Hudson-Taylor DE, et al. (1995) Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes. Cell 82: 101–110 Soma, D. D., Kassié, D., Sanou, S., Karama, F. B., Ouari, A., Mamai, W., Ouédraogo, G. A., Salem, G., Dabiré, R. K., & Fournet, F. (2018). Uneven malaria transmission in geographically distinct districts of Bobo-Dioulasso, Burkina Faso. Parasites & vectors, 11(1), 296. Spencer Valero, L. M., Ogun, S. A., Fleck, S. L., Ling, I. T., Scott-Finnigan, T. J., Blackman, M. J., & Holder, A. A. (1998). Passive immunization with antibodies against three distinct epitopes on Plasmodium yoelii merozoite surface protein 1 suppresses parasitemia. Infection and immunity, 66(8), 3925–3930. Spencer, H., Collins, W., Chin, W., & Skinner, J. (1979). The enzyme-linked immunosorbent assay (ELISA) for malaria I. The use of in vitro-cultured Plasmodium falciparum as antigen. Am J TropMed Hyg, 28, 927 – 932. Stevenson, M. M., Tam, M. F., & Nowotarski, M. (1990). Role of interferon-gamma and tumor necrosis factor in host resistance to Plasmodium chabaudi AS. Immunology letters, 25(1-3), 115–121. Stone, W.J.R., Campo, J.J., Ouédraogo, A.L. et al.(2018) Unravelling the immune signature of Plasmodium falciparum transmission-reducing immunity. Nat Commun 9, 558. Su XZ, Heatwole VM, Wertheimer SP, Guinet F, Herrfeldt JA, et al. (1995) The large, diverse gene family var encodes proteins involved in cytoadherence and antigenic 79 University of Ghana http://ugspace.ug.edu.gh variation of Plasmodium falciparum-infected erythrocytes. Cell 82: 89–100. Suh, K. N., Kain, K. C., & Keystone, J. S. (2004). Malaria. CMAJ : Canadian Medical Association journal = journal de l'Association medicale canadienne, 170(11), 1693–1702. Ta TH, Hisam S, Lanza M, Jiram AI, Ismail N, Rubio JM. The first case of a naturally acquired human infection with Plasmodium cynomolgi. Malar J. 2014 Feb 24;13:68. Takeuchi O, Akira S (2010) Pattern recognition receptors and inflammation. Cell 140: 805–820. Taylor, S. M., Cerami, C., & Fairhurst, R. M. (2013). Hemoglobinopathies: slicing the Gordian knot of Plasmodium falciparum malaria pathogenesis. PLoS pathogens, 9(5), e1003327. Taylor, S. M., Parobek, C. M., & Fairhurst, R. M. (2012). Haemoglobinopathies and the clinical epidemiology of malaria: a systematic review and meta-analysis. The Lancet. Infectious diseases, 12(6), 457–468. Taylor-Robinson, A. W., Phillips, R. S., Severn, A., Moncada, S., & Liew, F. Y. (1993). The role of TH1 and TH2 cells in a rodent malaria infection. Science (New York, N.Y.), 260(5116), 1931–1934. Trager, W., & Jensen, J. B. (1976). Human malaria parasites in continuous culture. Science (New York, N.Y.), 193(4254), 673–675. Traoré, D.F., Sagna, A.B., Adja, A.M. et al. (2019). Exploring the heterogeneity of human exposure to malaria vectors in an urban setting, Bouaké, Côte d'Ivoire, using an immuno-epidemiological biomarker. Malar J 18, 68 Trends in Microbiology, Volume 5, Issue 6,1997, Pages 213-215, 80 University of Ghana http://ugspace.ug.edu.gh Treutiger, C. J., Hedlund, I., Helmby, H., Carlson, J., Jepson, A., Twumasi, P., Kwiatkowski, D., Greenwood, B. M., & Wahlgren, M. (1992). Rosette formation in Plasmodium falciparum isolates and anti-rosette activity of sera from Gambians with cerebral or uncomplicated malaria. The American Journal of tropical medicine and hygiene, 46(5), 503–510. Tripathi AK, Sha W, Shulaev V, Stins MF, Sullivan DJ Jr (2009) Plasmodium falciparum- infected erythrocytes induce NF-kappaB regulated inflammatory pathways in human cerebral endothelium. Blood 114: 4243–4252. Troye-Blomberg M et al.(1999) Human gamma delta T cells that inhibit the in vitro growth of the asexual blood stages of the Plasmodium falciparum parasite express cytolytic and proinflammatory molecules. Scand J Immunol 50, 642–650 Troye-Blomberg, M., Romero, P., Patarroyo, M. E., Björkman, A., & Perlmann, P. (1984). Regulation of the immune response in Plasmodium falciparum malaria. III. Proliferative response to antigen in vitro and subset composition of T cells from patients with acute infection or from immune donors. Clinical and experimental immunology, 58(2), 380–387. Vallejo, A., Muniesa, A., Ferreira, C., & de Blas, I. (2013). New method to estimate the sample size for calculation of a proportion assuming a binomial distribution. Research in veterinary science, 95(2), 405–409. Villa P, Bigini P, Mennini T, Agnello D, Laragione T, et al. (2003) Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis. J Exp Med 198: 971–975. Voller, A., and O'Neill, P., (1971). Immunofluorescence method suitable for large-scale application to malaria. Bull. World Health Organ.45, 524–529 81 University of Ghana http://ugspace.ug.edu.gh Wagener FA, Eggert A, Boerman OC, Oyen WJ, Verhofstad A, et al. (2001) Heme is a potent inducer of inflammation in mice and is counteracted by heme oxygenase. Blood 98: 1802–1811. Walker, R. J. (2015). Multiplex immunoassays for hemoglobin, hemoglobin variants, and glycated forms. Google Patents, US20170176462A1 Wanaguru, M., Crosnier, C., Johnson, S., Rayner, J. C., & Wright, G. J. (2013). Biochemical analysis of the Plasmodium falciparum erythrocyte-binding antigen- 175 (EBA175)-glycophorin-A interaction: implications for vaccine design. The Journal of biological chemistry, 288(45), 32106–32117. Weatherall D. (2011). The inherited disorders of hemoglobin: an increasingly neglected global health burden. The Indian Journal of medical research, 134(4), 493–497. Weatherall, D. J., & Provan, A. B. (2000). Red cells I: inherited anemias. Lancet (London, England), 355(9210), 1169–1175. Weidanz, W. P., Brake, D. A., Cavacini, L. A., & Long, C. A. (1988). The protective role of T cells in immunity to malaria. Advances in experimental medicine and biology, 239, 99–111. WHO (1999). New Perspectives: Malaria Diagnosis. Report of joint WHO/USAID informal consultation 25–27 October 1999. Report No. WHO/CDS/RBM/2000.14. Williams TN, Weatherall DJ, Newbold CI (2002) The membrane characteristics of Plasmodium falciparum-infected and -uninfected heterozygous alpha(0)thalassaemic erythrocytes. Br J Haematol 118: 663–670. Williams, T. N., & Obaro, S. K. (2011). Sickle cell disease and malaria morbidity: a tale with two tails. Trends in parasitology, 27(7), 315–320. Wipasa, J., Suphavilai, C., Okell, L. C., Cook, J., Corran, P. H., Thaikla, K., Liewsaree, 82 University of Ghana http://ugspace.ug.edu.gh W., Riley, E. M., & Hafalla, J. C. (2010). Long-lived antibody and B Cell memory responses to the human malaria parasites, Plasmodium falciparum and Plasmodium vivax. PLoS pathogens, 6(2), e1000770. World Health Organization. World Malaria Report 2019. Geneva, Switzerland. http://www.who.int/malaria/publications Ya-Umphan, P., Cerqueira, D., Parker, D. M., Cottrell, G., Poinsignon, A., Remoue, F., Brengues, C., Chareonviriyaphap, T., Nosten, F., & Corbel, V. (2017). Use of an Anopheles Salivary Biomarker to Assess Malaria Transmission Risk Along the Thailand-Myanmar Border. The Journal of infectious diseases, 215(3), 396–404. 83 University of Ghana http://ugspace.ug.edu.gh APPENDICE Appendix 1 ELISA PLATE FORMATS FOR ELISA 1 2 3 4 5 6 7 8 9 10 11 12 A Std IgG Std IgG S_01Aa S_01Ab S_09Aa S_09Ab S_17Aa S_17Ab S_25Aa S_25Ab S_33Aa S_33Ab 1000ng/ml 1000ng/ml B 333.3 333.3 S_02Aa S_02Ab S_10Aa S_10Ab S_18Aa S_18Ab S_26Aa S_26Ab S_34Aa S_34Ab ng/ml ng/ml C 111.1 111.1 S_03Aa S_03Ab S_11Aa S_11Ab S_19Aa S_19Ab S_27Aa S_27Ab S_35Aa S_35Ab ng/ml ng/ml D 37.0 ng/ml 37.0 S_04Aa S_04Ab S_12Aa S_12Ab S_20Aa S_20Ab S_28Aa S_28Ab S_36Aa S_36Ab ng/ml E 12.3 ng/ml 12.3 S_05Aa S_05Ab S_13Aa S_13Ab S_21Aa S_21Ab S_29Aa S_29Ab S_37Aa S_37Ab ng/ml F 4.1 ng/ml 4.1 S_06Aa S_06Ab S_14Aa S_14Ab S_22Aa S_22Ab S_30Aa S_30Ab PC01 PC02 ng/ml G 1.4 ng/ml 1.4 S_07Aa S_07Ab S_15Aa S_15Ab S_23Aa S_23Ab S_31Aa S_31Ab NC01 NC02 ng/ml H 0.5 ng/ml 0.5 S_08Aa S_08Ab S_16Aa S_16Ab S_24Aa S_24Ab S_32Aa S_32Ab Blank01 Blank02 ng/ml 84 University of Ghana http://ugspace.ug.edu.gh Appendix 3 Preparation of buffers and media 0.05 M carbonate/bicarbonate buffer, pH 9.2, Sodium hydrogen carbonate (2.93 g) and sodium carbonate (1.59 g) in 1 liter of distilled water and add 1ml of methyl red from (1%) stock solution 1X PBS One tablet of PBS into 500ml of distilled water. Stir until it dissolves using a magnetic stirrer Washing buffer: PBST (PBS + 0,1% Tween) For every 1000ml of PBS, add 1ml of Tween and stir using a magnetic stirrer till it foams Blocking buffer: 3 % milk powder in PBST For every 100ml of PBST, add 3g of skimmed milk powder Sample Incubation buffer: 1 % milk powder in PBST For every 100ml of PBST, add 0.02g of sodium azide and 1g of skimmed milk powder. Stir to dissolve. Preparation of standard for IgG 1) In tube 1, add 1.7ml of coating buffer. 2) In tubes 2 – 8, add 1 ml of coating buffer (carbonate or PBS). 3) Transfer 1.7 µl of the stock IgG solution into tube one and mix well. 4) Transfer 0.5 ml from tube one into tube 2, and mix well. 5) Transfer 0.5 ml from tube two into tube 3, and mix well. Follow the order of step 5 up to tube 8. 0.2M H2SO4 1) For every 500ml of distilled water, add 1.15ml of conc H2SO4 85 University of Ghana http://ugspace.ug.edu.gh Appendix 4 Fig 1: Comparison of the IgG antibodies for gSG6-P1 levels in malaria microscopy positive and negative individuals in the Ashanti region. Fig 2: Comparison of the IgG antibodies for gSG6-P1 levels in malaria microscopy positive and negative individuals at the Brong Ahafo region. 86 University of Ghana http://ugspace.ug.edu.gh Fig 3: Comparison of the IgG antibodies for gSG6-P1 levels in malaria microscopy positive and negative individuals in the Central region. Fig 4: Comparison of the IgG antibodies for gSG6-P1 levels in malaria microscopy positive and negative individuals in the Eastern region. 87 University of Ghana http://ugspace.ug.edu.gh Fig 5: Comparison of the IgG antibodies for gSG6-P1 levels in malaria microscopy positive and negative individuals in the Greater Accra region. Fig 6: Comparison of the IgG antibodies for gSG6-P1 levels in malaria microscopy positive and negative individuals in the Northern region. 88 University of Ghana http://ugspace.ug.edu.gh Fig 7: Comparison of the IgG antibodies for gSG6-P1 levels in malaria microscopy positive and negative individuals in the Upper East region. Fig 8: Comparison of the IgG antibodies for gSG6-P1 levels in malaria microscopy positive and negative individuals at the Volta Region. 89 University of Ghana http://ugspace.ug.edu.gh Fig 9: Comparison of the IgG antibodies for gSG6-P1 levels in malaria microscopy positive and negative individuals in the Western Region. Fig 10: Comparison of the IgG antibodies for gSG6-P1 levels in malaria microscopy positive and negative individuals in the Upper West region 90 University of Ghana http://ugspace.ug.edu.gh Appendix 5 Fig 11: Comparison of the IgG antibodies for Pfs230 levels in malaria microscopy positive and negative individuals in the Ashanti region. Fig 12: Comparison of the IgG antibodies for Pfs230 levels in malaria microscopy positive and negative individuals at the Brong Ahafo region. 91 University of Ghana http://ugspace.ug.edu.gh Fig 13: Comparison of the IgG antibodies for Pfs230 levels in malaria microscopy positive and negative individuals in the Central region. Fig 14: Comparison of the IgG antibodies for Pfs230 levels in malaria microscopy positive and negative individuals in the Eastern region. 92 University of Ghana http://ugspace.ug.edu.gh Fig 15: Comparison of the IgG antibodies for Pfs230 levels in malaria microscopy positive and negative individuals in the Greater Accra region. Fig 16: Comparison of the IgG antibodies for Pfs230 levels in malaria microscopy positive and negative individuals in the Northern region 93 University of Ghana http://ugspace.ug.edu.gh Fig 17: Comparison of the IgG antibodies for Pfs230 levels in malaria microscopy positive and negative individuals in the Upper East region. Fig 18: Comparison of the IgG antibodies for Pfs230 levels in malaria microscopy positive and negative individuals in the Volta Region 94 University of Ghana http://ugspace.ug.edu.gh Fig 19: Comparison of the IgG antibodies for Pfs230 levels in malaria microscopy positive and negative individuals in the Upper West region. Fig 20: Comparison of the IgG antibodies for Pfs230 levels in malaria microscopy positive and negative individuals in the Western region. 95 University of Ghana http://ugspace.ug.edu.gh Appendix 6 Fig 21: Comparison of the IgG antibodies for EBA 175 R3 levels in malaria microscopy positive and negative individuals in the Ashanti region. Fig 22: Comparison of the IgG antibodies for EBA 175 R3 levels in malaria microscopy positive and negative individuals at the Brong Ahafo region. 96 University of Ghana http://ugspace.ug.edu.gh Fig 23: Comparison of the IgG antibodies for EBA 175 R3 levels in malaria microscopy positive and negative individuals in the Central region. Fig 24: Comparison of the IgG antibodies for EBA 175 R3 levels in malaria microscopy positive and negative individuals in the Eastern region. 97 University of Ghana http://ugspace.ug.edu.gh Fig 25: Comparison of the IgG antibodies for EBA 175 R3 levels in malaria microscopy positive and negative individuals in the Greater Accra region. Fig 26: Comparison of the IgG antibodies for EBA 175 R3 levels in malaria microscopy positive and negative individuals in the Northern region. 98 University of Ghana http://ugspace.ug.edu.gh Fig 27: Comparison of the IgG antibodies for EBA 175 R3 levels in malaria microscopy positive and negative individuals in the Upper East region. Fig 28: Comparison of the IgG antibodies for EBA 175 R3 levels in malaria microscopy positive and negative individuals in the Upper West region. 99 University of Ghana http://ugspace.ug.edu.gh Fig 29: Comparison of the IgG antibodies for EBA 175 R3 levels in malaria microscopy positive and negative individuals at the Volta Region. Fig 30: Comparison of the IgG antibodies for EBA 175 R3 levels in malaria microscopy positive and negative individuals in the Western region. 100 University of Ghana http://ugspace.ug.edu.gh Appendix 5 CLASSIFICATION OF HB PHENOTYPE Hb Hb A Hb F Hb S Hb E Hb A2 Hb C Hb D Hb H Phenotype AA > 95 < 3.5 AS > 45 < 10 ≤ 45 < 3.5 AC SS < 25 80 - 95 < 7 SC < 10 41 - 55 < 4 40 - 50 SD <10 ≈ 40 < 10 ≈ 50 S/β+ 15 - 45 < 25 45 - 75 < 6 SS/S-HPFH > 25 60 - 75 < 3.5 S/HIGH A2 < 20 40 - 70 20 - 45 Sickle Cell Syndromes By Maureen Achebe Edited by Edward J Benz Jr., Nancy Berliner, Fred J. Schiffman Publisher: Cambridge University Press pp 66 - 75 DOI: https://doi.org/10.1017/9781108586900.011 101