THE ROLE OF CIRCULATING ENDOTHELIAL PROGENITOR CELLS (CEPCs) AND OTHER BIOMARKERS IN THE PATHOGENESIS OF CEREBRAL MALARIA BY DANIEL ODURO 10048663 THIS DISSERTATION IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF PhD ANIMAL BIOLOGY DEGREE DECEMBER 2015 University of Ghana http://ugspace.ug.edu.gh ii DECLARATION I do declare that this thesis except for the references to other persons’ investigations which I have duly acknowledged, is the result of my own original research and that it has neither in whole nor in part been submitted for another degree anywhere. This work was done under the supervision of the persons mentioned below. Daniel Oduro (Candidate) Date Professor Ben Gyan (Principal Supervisor) Date Professor Dominic A. Edoh Supervisor Date Dr. Kwadwo Asamoah Kusi (Supervisor) Date University of Ghana http://ugspace.ug.edu.gh iii DEDICATION To my wife, Mrs. Salomey Oduro and my kids; Maame Afua, Nana Akua and Daniel (Jnr). Also to my parents, siblings and entire staff of Immunology Department, NMIMR especially Prof. Ben Gyan. University of Ghana http://ugspace.ug.edu.gh iv ACKNOWLEDGEMENT My acknowledgement goes to the National Institute of Health (NIH, U.S.A) and Noguchi Memorial Institute for Medical Research (NMIMR), EPCmal Study for funding this research. My appreciation goes to my supervisors, Prof. Ben Gyan and Dr. Asamoah Kusi both at the Noguchi Memorial Institute for Medical Research, and Prof. Dominic Edoh for their invaluable assistance. I am very grateful to Dr. Linnie Golightly of the Infectious Disease Division of Weill Cornell Medical School, New York. Mr. John Kweku Amissah Tetteh of the Immunology Department of NMIMR is well appreciated for training in the Laboratory and in Flow cytometric techniques. Staff of the collaborating Hospitals, Princess Marie Louis Hospital (Children’s Hospital), Ridge Hospital, La General Hospital, LEKMA Hospital and Tema General Hospital are so much appreciated. Osu Home School, Hayward preparatory school, LEKMA Northern and Southern cluster of Schools are well appreciated for their collaboration. I am particularly indebted to these institutions for the training I went through: Noguchi Memorial Institute for Medical Research, University of Ghana, Weill Cornell Medical College of Cornell University, Albert Einstein Medical College of Yeshiva University, New York. Highly acknowledged are the contributions of Dr. Michael Ofori, Prof. Daniel Dodoo both Senior Research Fellows at NMIMR, Dr. Linda Amoah and Dr. Bright Adu, Research Fellows at the Immunology Dept. NMIMR. University of Ghana http://ugspace.ug.edu.gh v This study would not have been possible without the contributions form the following members of the EPC team: Ms Hannah Tetteh, Mr. Eric Kyei-Baafour, Mrs. Dorotheah Obiri, Mr. Thomas Kwame Addison, Mr. Emmanuel Kakra Dickson, Mr. Gideon Obeng, Mrs. Amma Akorful- Andam, Mrs. Helena Lamptey, Ms. Dorothy Anum, Mr. Jones Amponsah and Ms. Sophia Ayia- Ampah, University of Ghana http://ugspace.ug.edu.gh vi TABLE OF CONTENTS Contents DECLARATION ............................................................................................................................................... ii DEDICATION................................................................................................................................................. iii ACKNOWLEDGEMENT ................................................................................................................................. iv TABLE OF CONTENTS ................................................................................................................................... vi LIST OF FIGURES .......................................................................................................................................... xi LIST OF TABLES ...........................................................................................................................................xiii ABBREVIATIONS ......................................................................................................................................... xiv ABSTRACT ................................................................................................................................................. xviii CHAPTER ONE ............................................................................................................................................. 20 1 INTRODUCTION ....................................................................................................................................... 20 1.1 Background ..................................................................................................................................... 20 1.2 Justification and Objectives ........................................................................................................... 24 1.2.1 Justification ................................................................................................................................ 24 1.2.2 Aims and Objectives .................................................................................................................. 26 CHAPTER TWO ............................................................................................................................................ 28 2 LITERATURE REVIEW ............................................................................................................................... 28 2.1 Socio-economic Burden of Malaria ............................................................................................... 28 2.1.1 Global malaria burden ................................................................................................................ 28 2.1.2 Malaria burden in Ghana ............................................................................................................ 29 University of Ghana http://ugspace.ug.edu.gh vii 2.3 Malaria Infection ............................................................................................................................ 30 2.3.1 Malaria parasite .......................................................................................................................... 30 2.3.2 Malarial life cycle ...................................................................................................................... 31 2.4 Severe Malaria ................................................................................................................................ 33 2.5 Cerebral Malaria ............................................................................................................................ 35 2. 5.1 Pathogenesis of cerebral malaria ............................................................................................... 37 2.5.2 Sequestration in CM................................................................................................................... 38 2.5.3 Role of cytokines and immune mediators in CM ....................................................................... 40 2. 5.4 Endothelial activation and blood-brain barrier integrity in CM ................................................ 42 2.5.5 Role of Angiopoietin 1 and 2 in CM .......................................................................................... 44 2.5.6 Role of thrombomodulin in CM ................................................................................................. 47 2.5.7 Role of endothelial protein C receptor (EPCR) in CM .............................................................. 49 2.6 HRP 2 and Cerebral Malaria ......................................................................................................... 51 2.7.1 Endothelial progenitor cell (EPC) and endothelial repair .......................................................... 53 2.7.2 Circulating endothelial cells (CEC) ........................................................................................... 55 2.7.3 CEC and EPC identification and quantification ......................................................................... 57 CHAPTER THREE .......................................................................................................................................... 60 3 MATERIALS AND METHODS .................................................................................................................... 60 3.1 Chemicals, Reagents and Equipment ............................................................................................ 60 3.2 Study Sites........................................................................................................................................ 60 University of Ghana http://ugspace.ug.edu.gh viii 3.3 Study Design .................................................................................................................................... 62 3.4 Ethical clearance ............................................................................................................................. 64 3.5 Inclusion/Exclusion Criteria .......................................................................................................... 64 3.5.1 Specific inclusion criteria ........................................................................................................... 64 3.5.2 Specific exclusion criteria .......................................................................................................... 65 3.6 Blood Sample Collection ................................................................................................................. 65 3.7 Sample Processing Time and Storage ........................................................................................... 66 3.8 Laboratory Evaluations/Assays ..................................................................................................... 67 3.8.1 Parasitology ................................................................................................................................ 67 3.8.2 Haematology .............................................................................................................................. 69 3.8.3 Bacteraemia evaluation .............................................................................................................. 69 3.8.4 Flow cytometric analysis ........................................................................................................... 70 3.8.5 Quantification of cEPC and CEC in whole blood ...................................................................... 72 3.8.6 Evaluation of chemokine/protease and endothelial biomarkers ................................................. 74 3.8.7 Statistical Analysis ..................................................................................................................... 75 CHAPTER FOUR ........................................................................................................................................... 76 4 RESULTS ................................................................................................................................................... 76 4.1 Background and Demographics of Study Participants ............................................................... 76 4.2 Stability of cells in C-C BCT .......................................................................................................... 79 4.2.1 Stability of leukocyte populations in C-C BCT preservative ......................................................... 79 University of Ghana http://ugspace.ug.edu.gh ix 4.2.2 Stability of EPCs in C-C BCT preservative ............................................................................... 79 4.2.3 Stability of CECs in C-C BCT preservative .............................................................................. 80 4.2.4 Stability of common immune markers in C-C BCT .................................................................. 81 4.2.5 Stability of endothelial and other markers in C-C BCT ............................................................. 82 4.3 Levels of cEPCs and CECs in the Study Groups ......................................................................... 85 4.3.1 cEPC levels in different groups at baseline ................................................................................ 85 4.3.2 Time course estimation of cEPC levels in different groups ....................................................... 86 4.3.3 CEC levels in different groups at initial presentation ................................................................ 87 4.3.4 Time course evaluation of CECs ............................................................................................... 88 4.4 Plasma levels of Ang-1 and Ang-2 ................................................................................................. 89 4.5 Levels of Endothelial Receptors..................................................................................................... 91 4.5.1 Baseline levels of soluble EPCR in the study groups ................................................................ 91 4.5.2 Time course levels of soluble EPCR in the study groups .......................................................... 92 4.5.3 Baseline levels of soluble TM in the study groups .................................................................... 92 4.5.4 Time course levels of soluble TM in the study groups .............................................................. 94 4.6 HRP2 levels ...................................................................................................................................... 95 4.6.1 Baseline levels of HRP 2 in the study groups ............................................................................ 95 4.6.2 Time course levels of HRP2 in the study groups ....................................................................... 96 4.7 Protease/Chemokine Levels ........................................................................................................... 97 4.7.1 Baseline levels of MMP9 in the study groups ............................................................................... 97 University of Ghana http://ugspace.ug.edu.gh x 4.7.2 Time course levels of MMP9 in the study groups ..................................................................... 98 4.7.3 Baseline levels of SDF-1 in the study groups ............................................................................ 99 4.7.4 Time course levels of SDF-1 in the study groups .................................................................... 100 CHAPTER FIVE ........................................................................................................................................... 101 5 DISCUSSION, CONCLUSION AND RECOMMENDATIONS ...................................................................... 101 5.1 Discussion....................................................................................................................................... 101 5.2 Conclusion .......................................................................................................................................... 113 5.3 Recommendations ......................................................................................................................... 114 REFERENCES .............................................................................................................................................. 116 APPENDICES .............................................................................................................................................. 143 Appendix 1: Buffers and Reagents .................................................................................................... 143 Appendix 2A: Consent for Children with Malaria .......................................................................... 144 Appendix 2B: Consent for Healthy Controls ................................................................................... 149 Appendix 3A: Assessment Forms/Questionnaires for Cerebral Malaria Cohort ......................... 154 Appendix 3B: Assessment Forms/Questionnaires for Uncomplicated Malaria Cohort ............... 162 Appendix 3C: Assessment Form/Questionnaire for Healthy Volunteer Cohort ........................... 167 University of Ghana http://ugspace.ug.edu.gh xi LIST OF FIGURES Figure 1. 1 Model of the development and resolution of cerebral malaria ................................................. 27 Figure 2. 1 Worldwide Malaria death rate. ................................................................................................. 30 Figure 2. 2 Simplified malaria lifecycle within the mammalian host ......................................................... 32 Figure 2. 3 Diagram showing proposed mechanical hypothesis of the pathogenesis of cerebral malaria .. 38 Figure 2. 4 Coronal section of the brain showing iRBC sequestration and microvascular thrombosis in fatal cerebral malaria. .......................................................................................................................................... 40 Figure 2. 5 Proposed mechanism of the role of Angiopoietins in immunopathogenesis of CM. ............... 45 Figure 2. 6 Role of EPCR in CM ................................................................................................................ 51 Figure 2. 7 Circulating EPC contribute to endothelial repair ..................................................................... 54 Figure 2. 8 Schematic representation of endothelial damage ..................................................................... 55 Figure 3. 1 Map of Greater Accra region showing the location of the study sites (red) ............................. 61 Figure 3. 2 Schematic presentation of the study design .............................................................................. 63 Figure 3. 3 Thick and thin blood film ......................................................................................................... 68 Figure 3. 4 Flow cytometer (FACS Calibur) .............................................................................................. 70 Figure 3. 5 cEPCs gating strategy and estimation....................................................................................... 72 Figure 3. 6 CEC gating strategy and estimation. ........................................................................................ 73 Figure 4. 1 Leukocyte populations in whole blood preserved in C-C BCT preservative and EDTA anticoagulant over a seven day period. ....................................................................................................... 79 Figure 4. 2 Percentage of cEPC at different time points in whole blood preserved in C-C BCT. .............. 80 Figure 4. 3 Percentage frequency of CEC at different time points in whole blood preserved EDTA anticoagulant and in C-C BCT.. .................................................................................................................. 81 Figure 4. 4 Percentage frequencies of some immune marker at different time points in whole blood preserved in C-C BCT. ............................................................................................................................... 82 University of Ghana http://ugspace.ug.edu.gh xii Figure 4. 5 Percentage cell count of individual cell expressing EPC, CEC, haematopoietic and leukocyte receptors in whole blood preserved in Cyto-chex BCT at 4oC for seven days. .......................................... 84 Figure 4. 6 cEPCs levels at initial evaluation in the study groups. ............................................................. 85 Figure 4. 7 Time course cEPC levels in study the groups. ......................................................................... 86 Figure 4. 8 Percentage CECs at initial evaluation in the study groups ....................................................... 87 Figure 4. 9 Time course CEC levels in the study groups ............................................................................ 88 Figure 4. 10 Soluble EPCR levels in study the groups ............................................................................... 91 Figure 4. 11 Levels of soluble EPCR at different time points in the study groups ..................................... 92 Figure 4. 12 Soluble TM levels in the study groups ................................................................................... 93 Figure 4. 13 Levels of soluble TM at different time points in the study groups ......................................... 94 Figure 4. 14 HRP2 levels in the study groups at baseline ........................................................................... 95 Figure 4. 15 Time course HRP2 levels in the study groups ........................................................................ 96 Figure 4. 16 MMP9 levels in the study groups at baseline ......................................................................... 97 Figure 4. 17 Time course MMP9 levels in the study groups ...................................................................... 98 Figure 4. 18 SDF-1 levels in the study groups at baseline .......................................................................... 99 Figure 4. 19 Time course SDF-1 levels in the study groups ..................................................................... 100 University of Ghana http://ugspace.ug.edu.gh xiii LIST OF TABLES Table 2. 1 Definition of EPCs and CECs .................................................................................................... 59 Table 3. 1 Monoclonal antibodies against endothelial receptors ................................................................ 71 Table 4. 1 General Characteristics of study participants ............................................................................. 78 Table 4. 2 Time course Plasma levels of Ang-1 and -2 in the study groups...................................... 90 University of Ghana http://ugspace.ug.edu.gh xiv ABBREVIATIONS Ang-1 Angiopoietin-1 Ang-2 Angiopoietin-2 APC Activated Protein C APC Allophycocyanin BBB Blood Brain Barrier BCS Blantyre Coma Score BD Becton Dickinson BSA Bovine Serum Albumin CD Cluster of Differentiation C-C BCT Cyto-Chex Blood Collection Tube CECs Circulating Endothelial Cells CEPCs Circulating Endothelial Progenitor cells CI Confidence Interval CNS Central Nervous System CM Cerebral Malaria CSF Cerebrospinal Fluid ECs Endothelial Cells EDTA Ethylenediamine tetra acetic acid ELISA Enzyme-Linked Immunosorbent Assay EPCs Endothelial Progenitor Cells EPCR Endothelial Protein C Receptor FACS Fluorescence –Activated Cell Sorting University of Ghana http://ugspace.ug.edu.gh xv FITC Fluorescein Isothiocyanate GDP Gross Domestic Product Hb Haemoglobin HC Healthy Control HCT Haematocrit HIV Human Immunodeficiency-Virus HRP2 Histidine Rich Protein 2 HSCs Hematopoietic Stem Cells ICAM-1 Intercellular Adhesion Molecule-1 IFN-ɣ Interferon-ɣ Ig Immunoglobulin IL Interleukin IRB Institutional Review Board iRBC Infected Red Blood Cell ITNs Insecticide-Treated Nets KDR Kinase-insert Domain Receptor LEKMA Ledzokuku Krowoh Municipal Authority MAb Monoclonal Antibody MCH Mean Corpuscular Haemoglobin MCHC Mean Corpuscular Haemoglobin Content MCV Mean Corpuscular Volume MMP-9 Matrix Metalloproteinase-9 NGS Normal Goat Serum University of Ghana http://ugspace.ug.edu.gh xvi NMIMR Noguchi Memorial Institute for Medical Research NO Nitric Oxide OPD Out Patient Department PB Peripheral Blood PBS Phosphate Buffered Saline PC Protein C PE Phycoerythrin PECAM-1 Platelet Endothelial Cell Adhesion Molecule PerCP Peridinin-Chlorophyll-Protein Complex PfEMP1 Plasmodium falciparum Erythrocyte Membrane Protein 1 PfGPI Plasmodium falciparum Glycosylphosphatidylinositol RBM Roll Back Malaria RBC Red Blood Cell RDT Rapid Diagnostic Test SAH Sub-Arachnoid Haemorrhage SDF-1 Stromal Cell-Derived Growth Factor-1 sEPCR Soluble Endothelial Protein C Receptor SMA Severe Malaria Anaemia sTM soluble Thrombomodulin TGF Transforming Growth factor TM Thrombomodulin TSP Thrombospondin TNF- α Tumor Necrosis Factor-α University of Ghana http://ugspace.ug.edu.gh xvii UCB Umbilical Cord Blood UM Uncomplicated Malaria UNICEF United Nations International Children’s Emergency Fund VCAM-1 Vascular Cell Adhesion Molecule VEGF Vascular Endothelial Growth Factor VEGFR-1 Vascular Endothelial Growth Factor Receptor 1 VEGFR-2 Vascular Endothelial Growth Factor Receptor 2 VWF Von Willebrand Factor VWFpp Von Willebrand Factor propeptides WBC White Blood Cell WHO World Health Organization WPB Weibel-Palade Bodies University of Ghana http://ugspace.ug.edu.gh xviii ABSTRACT Cerebral malaria (CM) is known to be the most severe complication of Plasmodium falciparum infection. Despite effective anti-parasitic treatment, it remains a major cause of morbidity and mortality in infected children. Sequestration of infected red blood cells in the brain microvasculature, occlusion of blood flow, activation and subsequent damage of the endothelium are hallmarks of the pathogenesis of CM. This study sought to quantify and compare time course levels of cEPC and other biomarkers of endothelial injury and repair in Ghanaian children between the ages of 2 and 12 years with cerebral malaria, uncomplicated malaria as well as uninfected healthy controls. Participants with malaria were recruited from five main referral hospitals in Accra, Ghana and healthy uninfected controls from community schools around the hospitals. Matrix metalloproteinase-9 (MMP9), stromal cell-derived growth factor 1 (SDF-1), Angiopoietin 1 and 2, Endothelial protein C receptor (EPCR), thrombomodulin (TM) and Histidine Rich Protein 2 (HRP2) levels were also determined by enzyme-linked immunosorbent assay (ELISA) at the same time points in all study participants. Stability of cEPCs and CECs were assessed in whole blood stored in the cell preservative Cyto Chex BCT (C-C BCT) at 4oC and compared with that stored in ethylenediamine tetra acetic acid (EDTA) antocoagulant at baseline. CM patients showed a baseline mean percentage cEPC of 0.042% which increases to 0.117% at recovery from coma. cEPC levels in uncomplicated malaria (UM) and uninfected healthy controls (HC) were 0.147% and 0.83% respectively at baseline with no significant difference in time course. CEC levels in CM was higher (0.003%) at initial presentation compared with uncomplicated (0.001%) and uninfected healthy controls (0.0009%). However, CEC levels in UM patients spiked (0.009%) at day 7. HC maintained a low CEC levels in time course. SDF-1 levels remained unchanged in all study groups in time course whiles MMP9 levels were higher in UM patient compared with CM University of Ghana http://ugspace.ug.edu.gh xix and HC at baseline. Angiopoietin 1 (Ang-1) levels were higher (5960pg/ml) in CM compared to UM (4041pg/ml) and HC (4909pg/ml) though not significant. TM, EPCR and HRP2 level was highest in CM compared with other groups at baseline. The study has shown that cEPCs and the mediators associated with their release and migration are very critical in the resolution of coma in children and has placed CM within the context of current paradigms of microvascular repair, offering strategies that could predict who is at risk of developing CM. Therapies that mobilize and improve cEPC function will therefore be of immense utility in the prevention and treatment of CM. University of Ghana http://ugspace.ug.edu.gh 20 CHAPTER ONE 1 INTRODUCTION 1.1 Background Malaria is a major global health problem that poses enormous burden on mankind, both socially and economically (Rénia et al., 2012). A report from World Health Organization (WHO) in 2014 shows that there were an estimated 198 million episodes of malaria worldwide in 2013 from which 584,000 resulted in death. Ninety percent (90%) of these deaths were recorded in Africa. It is also estimated that nearly a quarter of all childhood deaths are caused by malaria (Miller et al., 2013). Out of about 500 million clinical malaria cases that are reported each year, one percent of these cases may become complicated and develop into severe malaria (Idro et al., 2010). In some cases, however, the disease becomes so severe and may lead to death. Malaria is known to be caused by five different species of the Plasmodium parasite of which Plasmodium falciparum is responsible for nearly all of the severe morbidity and mortality in malaria endemic areas (WHO, 2000). About 91% of malaria cases worldwide are caused by P. falciparum, with the majority (86%) occurring in the African region (WHO, 2008). Cerebral malaria is known to be the deadliest form of severe malaria and probably amongst the most common non-traumatic encephalopathies in the world due to the fact that its case fatality rate is about 20% and about 7% of children who survive are left with permanent neurological disability, epilepsy or behavioural problems (Birbeck et al., 2010). CM collectively involves the clinical manifestations of P. falciparum malaria that induces changes in mental status and coma (Ozen et al., 2006). Malaria can occur in less than two weeks in non-immune individuals after a mosquito bite and CM may develop if not treated after 24 hours of onset of symptoms (WHO, 2000; WHO, University of Ghana http://ugspace.ug.edu.gh 21 2014a). Sequestration of infected red blood cells (iRBCs) in the brain microvasculature has been shown to be the hallmark of CM and the resultant damage to the vascular endothelium has been postulated as a major initiator of CM (Cooke et al., 2000; Silamut et al., 1999a; Taylor et al., 2004b; Weatherall et al., 2002). The ability to balance microvascular damage and repair may therefore be critical in the pathogenesis of cerebral malaria. Studies have shown that recovery from endothelial damage as seen in CM, is defined by a balance between the magnitude of microvascular damage and the capacity for repair (Hill et al., 2003). Repair of damaged endothelium can occur by migration and proliferation of surrounding mature endothelial cells (ECs). However, mature ECs are terminally differentiated cells with a low proliferative potential, and their capacity to substitute damaged endothelium is limited (Hristov et al., 2003a). Therefore, endothelial repair may need the support of other cell types such as bone marrow–derived endothelial progenitor cells (EPCs) which migrate to sites of damage and incorporate into the microvasculature (Lin et al., 2000; Rafii, 2000) to augment the local response which may be insufficient to repair extensive or chronic injury (Gyan et al., 2009). Some studies have shown EPCs to be capable of facilitating vascular repair in different ischaemic tissues (Medina et al., 2010). A cross-sectional evaluation of circulating EPC (cEPC) levels in cerebral malaria by Gyan et al. (2009) associated low levels of cEPCs with CM in African children, indicating the importance of cEPCs in microvascular repair in P. falciparum infection. The presence of circulating ECs (CECs) has been recognized as a useful marker of microvascular damage (Goon et al., 2006b). Acute vascular injury has been correlated with an increase in the number of CECs and bone marrow-derived cEPCs in the peripheral blood (Wu et al., 2007). University of Ghana http://ugspace.ug.edu.gh 22 Although rare in healthy individuals, increased CECs in peripheral blood reflects significant vascular damage and dysfunction (Goon et al., 2006b). Elevated levels of CECs in peripheral blood in CM patients could therefore be predictive of the endothelial damage. Microvascular damage induces the expression or activation of a series of molecules such as stromal cell-derived growth factor 1 (SDF-1) and the matrix metalloproteinase-9 (MMP-9) which mediate the mobilization and release of EPCs from the bone marrow (Adams et al., 2004; Carmeliet and Collen, 2000; Heissig et al., 2002; Hristov et al., 2003b; Ruhrberg, 2003; Szmitko et al., 2003; Urbich and Dimmeler, 2004). Elevated levels of SDF-1 in acute malaria infection could determine to a larger extent, the mobilization and release of EPCs from the bone marrow and subsequent repair of damaged endothelium (Gyan et al., 2009). These chemokine/proteases could therefore play a major role in the pathogenesis of CM. Other endothelial mediators such as the Angiopoietins (Ang-1 and Ang-2) are known to be important regulators of vascular structure and function, and are hallmark indicators of vascular injury (Chittiboina et al., 2013). These proteins have shown promise as targets in the treatment of diseases such as traumatic brain injury, sub-arachnoid haemorrhage (SAH) and sepsis (Chittiboina et al., 2013; Parikh, 2013). Angiopoietins have also been shown to discriminate cerebral malaria and severe, non-cerebral, malaria from uncomplicated malaria (Conroy et al., 2009; Lovegrove et al., 2009) indicating the importance of these mediators in the pathogenesis of severe malaria. Thrombomodulin (TM) and Endothelial Protein C Receptor (EPCR) are receptors expressed on endothelial cells and are essential components of the anticoagulant protein C pathway, an University of Ghana http://ugspace.ug.edu.gh 23 endothelial homeostatic signal critical in regulating coagulation, inflammation, endothelial barrier function, and neuro-protection (Esmon, 2000). These receptors are shed into peripheral blood when the endothelium is inflamed and increased levels of soluble forms of these receptors in peripheral blood have been shown in cerebral malaria (Boehme et al., 1996; Moxon et al., 2013). Soluble TM and EPCR therefore show promise as predictors of cerebral malaria. Sequestration of asexual parasites in the brain microvasculature and other organs in the severe malaria host makes identification of peripheral parasitaemia difficult. Histidine Rich Protein 2 (HRP2), a protein produced by P. falciparum has been used as a diagnostic marker and to estimate parasite burden in severe malaria (Storm and Craig, 2014). Elevated levels of this protein has also been used to distinguish coma caused by CM and other infections (Kariuki and Newton, 2014). This study therefore aimed at characterizing the hosts’ response to P. falciparum induced microvascular damage and determine its relationship to the development of, and recovery from, cerebral malaria. This was done by longitudinally determining the levels of CECs, EPCs, chemokines/proteases (SDF-1 and MMP-9) associated with the release of EPCs and endothelial mediators (Angiopoietins), endothelial receptors (soluble TM and soluble EPCR) as well as P. falciparum parasite protein, HRP2, in children with cerebral malaria, uncomplicated malaria and uninfected healthy controls. EPCs by nature are very rare in peripheral blood (Duda et al., 2007) and their determination by flow cytometric analysis must be done immediately after collection and staining of fresh whole blood samples (NCCLS, 1998, as cited in (Schumacher and Burkhead, 2000)). The study also University of Ghana http://ugspace.ug.edu.gh 24 aimed at developing methods that would allow preservation of samples for delayed flow cytometric analysis and for the extension of this technique to biological samples from remote settings where flow cytometers are not readily available. 1.2 Justification and Objectives 1.2.1 Justification Cerebral malaria is the most severe neurological complication of infection with Plasmodium falciparum and is a major cause of child death in sub-Saharan Africa. Several theories have evolved to define the pathogenesis of cerebral malaria. However, the sequestration of infected red blood cells in the brain microvasculature, activation and subsequent damage of the endothelium are hallmarks (Cooke et al., 2000; Silamut et al., 1999a; Taylor et al., 2004b; Weatherall et al., 2002). Damaged microvasculature was believed to be repaired solely by the replication or migration of local preexisting vascular wall endothelial cells to sites of injury (Asahara et al., 1997; Lin et al., 2000; Rafii, 2000). It is now known that circulating bone marrow derived EPCs are involved in the repair of microvascular damage (Asahara et al., 1999). They augment the local response, which may be insufficient to repair extensive or chronic injury by replication or migration. Studies suggests that therapies and tests based on microvascular homeostasis are currently being aggressively developed by industry to treat and determine who is at risk for cardiovascular events, stroke, asthma and ischemic disease, and might be of utility in cerebral malaria. The capability of cEPCs as biomarkers and their use as therapeutic agents for microvascular repair have shown promise in cardiovascular disease (Timmermans et al., 2009). University of Ghana http://ugspace.ug.edu.gh 25 Gyan et al (2009) associated cerebral malaria with low levels of cEPCs, thereby placing cerebral malaria pathogenesis within the context of the current paradigms of microvascular homeostasis. Their study postulated that an increase in cEPC levels could correlate with recovery from cerebral malaria. The body’s ability to elevate peripheral levels of cEPCs could therefore be predictive of recovery from CM. Several molecules have been implicated in the loss of endothelial integrity and by extension also implicated in the pathogenesis of cerebral malaria. These molecules may include stromal cell derived growth factor 1 (SDF-1) and the matrix metalloproteinase-9 (MMP-9) which mediate the mobilization and release of cEPCs (Adams et al., 2004; Carmeliet and Collen, 2000; Heissig et al., 2002; Hristov et al., 2003b; Ruhrberg, 2003; Szmitko et al., 2003; Urbich and Dimmeler, 2004), endothelial mediators such as Angiopoietin 1 and 2 (Lovegrove et al., 2009) and endothelial receptors such as EPCR and TM (Moxon et al., 2014). The repair role of EPCs and the other endothelial mediators could help address some questions that have remained unanswered in the pathogenesis of CM: why is coma so rapidly reversible with treatment despite the large number of parasites in the brain of most patients? Why do some children recover quickly from coma while others die? This study therefore aimed at determining factors that affect the levels and function of cEPCs and CECs and their relation with the progression of uncomplicated malaria to cerebral malaria. This knowledge would be important in addressing some unanswered questions as stated above, predict University of Ghana http://ugspace.ug.edu.gh 26 who will develops cerebral malaria and explore the possibility of cEPCs as therapeutic agents for cerebral malaria and in the development of vaccines. 1.2.2 Aims and Objectives Generally the aim of the study was to firstly determine and compare the time course host response to microvascular damage in Ghanaian children with CM, UM and HC. An additional objective was to investigate the ability of Cyto-Chex BCT to maintain the viability of receptors expressed by circulating endothelial cells and their progenitors 1.2.2.1 Specific objectives 1. To determine and compare levels of cEPC/CEC in whole blood stored with EDTA and C- C BCT at 4oC over seven days. 2. To determine levels of cEPC/CEC in CM patients at initial clinical presentation and at recovery from coma as well as at days seven and fourteen post recovery. 3. To determine levels of cEPC/CEC in UM patients and uninfected healthy controls at initial presentation and at days seven and fourteen after initial presentation. 4. To compare cEPC/CEC levels within the patient groups and across the three study groups. 5. To measure and compare levels of chemokines/proteases (SDF-1, and MMP-9), endothelial mediators (Angiopoietin 1 and 2), endothelial receptors (EPCR and TM), and HRP 2 at all sampling time points in the three study groups. University of Ghana http://ugspace.ug.edu.gh 27 1.2.2.2 Study hypothesis The study hypothesized that: 1. Increasing levels of cEPCs and decreasing levels of CECs and chemokine/protease correlate with recovery from cerebral malaria as shown in Figure 1.1 below: Figure 1. 1 Model of the development and resolution of cerebral malaria 2. Lower Ang-2: Ang-1 ratio correlates with recovery from cerebral malaria. 3. Increased levels of sEPCR and sTM are associated with cerebral malaria. 4. Higher HRP2 levels is higher in true CM than uncomplicated malaria University of Ghana http://ugspace.ug.edu.gh 28 CHAPTER TWO 2 LITERATURE REVIEW 2.1 Socio-economic Burden of Malaria 2.1.1 Global malaria burden The World Malaria Report of 2014 shows that approximately 3.3 billion people were at risk of malaria around the world and 219 million cases are estimated to have occurred. Africa alone accounts for 89% of malaria cases and 91% of malaria deaths (WHO, 2014b; WHO, 2014c). In 2013, malaria alone caused an estimated 453 000 under-five deaths globally and an estimated 437 000 African children died before their fifth birthday due to malaria in the same year (WHO, 2014c). In high-risk areas, more than one malaria case occurs per 1000 population and with the estimated number of deaths in 2013, it indicates 1300 children deaths every day (WHO, 2014c). More than 90 countries are known to have ongoing malaria transmission and hence malaria is considered a public health problem (WHO, 2014b). Despite the considerable public health efforts directed at treatment and control worldwide, malaria remains the most important parasitic disease globally with almost one-quarter of the world‘s population at risk (Hay et al., 2009). In Ghana, despite several gains in malaria control initiatives worldwide, malaria situation remains high (between 25-65 death rate per 100,00 population) [Figure 2.1], with a prevalence of 67% of households reporting an episode of malaria every two weeks (Musah, 2013). Malaria inflicts serious negative impact on health and lays a heavy economic and social burden on families, communities and societies in the poorest countries of the world and has thus been tagged as a disease of poverty (Karunamoorthi, 2012). It is the major cause of repeated work absenteeism in endemic regions and this results in short and long term losses in productivity as the main University of Ghana http://ugspace.ug.edu.gh 29 transmission periods coincide with the peak agricultural and harvesting seasons (Karunamoorthi and Bekele, 2009). In regions where malaria thrives, jobs and school days are lost, productivity plummets and entire communities remain locked in an unbreakable cycle of disease and poverty (RBM, 2013). When it does not kill, the disease can lead to permanent neurological and cognitive damage in children, thus impeding education, reducing career opportunities and lowering productivity in adult age. The direct and indirect costs of malaria have been shown to be a major constraint to economic development with the direct costs being a combination of personal and public expenditures on both prevention and treatment of the disease. At the micro level the personal expenditures include individual or family spending on insecticide-treated nets (ITNs), doctors’ fees, anti-malarials, transport to health facilities, and support for the patient and an accompanying family member during hospital stays (RBM, 2003). At the macro level the economic burden of malaria is estimated at an annual reduction in economic growth of 1.3% for those African countries with the highest burden (WHO, 2009). An estimated 12 billion USD loss to the African continent’s Gross Domestic Product (GDP) annually as a result of malaria (RMB, 2013). 2.1.2 Malaria burden in Ghana In Ghana, malaria has been reported as the major cause of poverty and low productivity, accounting for about 32.5 % of all OPD attendances and 48.8 % of children under five years admissions in the country (Aregawi et al., 2009). It is generally believed that malaria is the cause of the highest loss of number of days of healthy life in Ghana although reliable information on the impact of malaria on labour productivity and the economy is absent (Asenso-Okyere and Dzator, 1997). University of Ghana http://ugspace.ug.edu.gh 30 Figure 2. 1 Worldwide Malaria death rate. Adapted from Factsheet on WHO Report, 2013. 2.3 Malaria Infection 2.3.1 Malaria parasite Malaria is caused by infection of red blood cells by the protozoan parasite Plasmodium. There are five species of Plasmodium that infect humans (falciparum, vivax, ovale, malariae and knowlesi). These are transmitted by over 30 species of anopheline mosquitoes. Plasmodium falciparum is known to be the most deadly among the five species and results in a number of different pathologies associated with specific organ systems. It is responsible for almost all the mortality from malaria and is the only species that appears to directly affect the central nervous system (CNS) causing neurologic deficits and cognitive sequelae. P. falciparum is also known to be the predominant species in sub-Saharan Africa where over 90% of all malaria deaths occur (WHO, 2012). Ghana University of Ghana http://ugspace.ug.edu.gh 31 A key feature of the biology of P. falciparum is its ability to cause infected red blood cells (iRBCs) to adhere to the linings of small blood vessels. Such sequestered parasites cause considerable obstruction to tissue perfusion. In addition, in severe malaria there may be marked reductions in the deformability of uninfected RBCs (Dondorp et al., 2008). The majority of malarial deaths in Africa occur in children under 5 years of age, as non-sterile immunity develops with increasing age and recurrent exposure to malaria (Marsh, 1992). It remains unclear why some children develop the severe manifestations of disease while others suffer only mild symptoms or remain asymptomatic (Rowe et al., 1995). 2.3.2 Malarial life cycle Even-though the pathophysiology and management of malaria are well understood there is still high mortality in children (Miller et al., 2002). Understanding the malaria disease process begins with an understanding of the complex life cycle of Plasmodium species. Figure 2.2 shows a simplified malaria life cycle in the mammalian host. University of Ghana http://ugspace.ug.edu.gh 32 Figure 2. 2 Simplified malaria lifecycle within the mammalian host. Adapted from Serghides et al. (2003). Trends Parasitol. 19(10): 461-9. When a mosquito injects motile sporozoites into the human blood stream during a blood meal, they invade the liver cells where they multiply and generate thousands of merozoites. These merozoites are then released into the blood stream and start to invade healthy red blood cells (RBCs). The iRBCs go through a sequence of three main developmental stages – ring, trophozoite and schizont, also known as the asexual multiplication cycle. After 48 hours this sequence ends in the bursting and releasing of 16–32 new merozoites from the iRBCs, and the cycle of invasion and infection starts again. University of Ghana http://ugspace.ug.edu.gh 33 Some intraerythrocytic parasites take a different developmental path and produce male and female gametocytes to begin the sexual stage of the malaria life cycle within the mosquito midgut. Disease in P. falciparum is related to the latter half of the erythrocytic cycle, where: i) schizonts express parasite proteins that mediate the cytoadherence of iRBCs in the microvasculature; and ii) schizonts rupture releasing infective merozoites and other parasite-derived bioactive products. 2.4 Severe Malaria Despite several breakthroughs in understanding Plasmodium biology, including the sequencing of the Plasmodium falciparum genome (Gardner et al., 2002) and efforts to eradicate the mosquito vector through widespread insecticide campaigns, people (mostly children) are still dying as a result of severe complications of malaria. Severe malaria is most commonly caused by infection with P. falciparum, although P. vivax and P. knowlesi can also cause severe disease. However, nearly all deaths from severe malaria result from infections with P. falciparum. The risk is increased if treatment of an uncomplicated attack of malaria caused by these parasites is delayed. Sometimes, however, especially in children, severe P. falciparum malaria may develop so rapidly that early treatment of uncomplicated malaria is not feasible (Michael and World Health Organization, 2000). In high-transmission areas, the risk for severe falciparum malaria is greatest among young children and visitors of any age from non- endemic areas. It is defined by clinical or laboratory evidence of vital organ dysfunction. Severe malaria can mimic many other diseases that are also common in malaria-endemic countries. The University of Ghana http://ugspace.ug.edu.gh 34 most important of these are central nervous system infections, septicaemia, severe pneumonia and typhoid fever. Severe malaria develops due to the fact that parasites sequester themselves in various organs including heart, lung, brain, liver, kidney, subcutaneous tissues and placenta (Miller et al., 2002). It has been shown that one or more of these organs can be affected with different levels of severity and this can be classified as neurologic and renal dysfunction, haematologic, cardiovascular, and respiratory dysfunction, as well as hepatic and metabolic dysfunction depending on the organ affected (Mohapatra and Das, 2009). These may be due to fact that, during the malaria cycle, iRBCs circulating in the blood stream begin to lose their deformability (MacPherson et al., 1985), thus becoming potential targets for filtering and destruction by the spleen. To avoid this, the parasite expresses and exports adhesive proteins to the surface of the host iRBC, causing the cell to stick to microvascular endothelial cells in different organs, thereby preventing its clearance by the spleen. Available evidence suggests that organ dysfunction and severe pathology follow the accumulation of iRBCs at high density in particular organs (MacPherson et al., 1985; Pongponratn et al., 1991). Adhesion is therefore believed to be one of the main causes of lethal complications resulting in cerebral malaria and placental malaria. Rapid expansion of iRBC mass, destruction of both infected and uninfected RBCs, microvascular obstruction as a result of parasite sequestration, and inflammatory processes are basic processes that combine to lead to reduced tissue perfusion in severe malaria (Miller et al., 2002). These, in turn, may lead to downstream events at the cellular level that further exacerbate the situation. University of Ghana http://ugspace.ug.edu.gh 35 The frequent presentations of severe falciparum malaria include cerebral malaria, metabolic malaria (hyperlactaemia, acidosis or respiratory distress) and severe anaemia (Dzeing-Ella et al., 2005; Miller et al., 2013). Most often than not, seizures, impaired consciousness, or metabolic acidosis presenting as respiratory distress or severe anaemia are usual manifestations of severe falciparum malaria in African children growing up in areas where malaria is endemic. African children rarely develop renal failure or pulmonary oedema (MacPherson et al., 1985). 2.5 Cerebral Malaria Most malaria-related deaths are associated with cerebral malaria (CM) and is arguably one of the most common non-traumatic encephalopathies in the world and remains a major cause of morbidity (Mishra and Newton, 2009). Cerebral malaria is considered the most severe form of malaria and is caused by infection with P. falciparum parasites (Miller et al., 2002). Plasmodium falciparum parasite is responsible for almost all the neurological complications associated with malaria, although P. vivax causes seizures in children, and is also associated with coma in both children and adults (Mishra and Newton, 2009). It is also considered one of the most dangerous diseases due to the fact that up to 30% of patients who develop cerebral malaria can die (Adams et al., 2002).. Children in sub-Saharan Africa account for 90% of CM-associated deaths (Dorovini-Zis et al., 2011) and it is estimated that over 785000 children younger than 9 years are affected every year in sub-Saharan Africa, (Newton and Krishna, 1998). Peak incidence is recorded in preschool University of Ghana http://ugspace.ug.edu.gh 36 children where approximately 575000 children are affected with cerebral malaria annually (Breman, 2001). The clinical hallmark of cerebral malaria is coma and this collectively involves the clinical manifestations of P. falciparum malaria that induces changes in mental status (Idro et al., 2005). The commonly accepted clinical definition of CM is the neurological syndrome with patients in unarousable coma (Newton et al., 1990). The World Health Organization also defines cerebral malaria as a clinical syndrome characterized by coma at least 1 hour after termination of a seizure or correction of hypoglycemia, asexual forms of Plasmodium falciparum parasites on peripheral blood smears, and no other cause to explain the coma (WHO, 2000) . In African children, cerebral malaria can occur in less than two weeks after a mosquito bite and coma develops suddenly with seizure onset often after 1–3 days of fever (Rénia et al., 2012). A few children develop coma after progressive weakness and prostration (Idro et al., 2010). Without treatment, cerebral malaria is invariably fatal. In children, parenteral anti-malarials (cinchonoids or artemisinin derivatives) are indicated, but even with this treatment, 15– 20% die (Idro et al., 2010; Kain et al., 1998). Although highly effective anti-malarial drugs are widely available, CM case fatality remains 15-20% globally. If a person is not treated, CM is fatal in 24 - 72 hours (Babikir, 2010). Earlier studies suggested that surviving patients fully recover (Muntendam et al., 1996) but over the past 20 years, it became clear that many children sustain significant brain injury; 11% are discharged with gross neurological deficits and these may include weakness, spasticity, blindness, University of Ghana http://ugspace.ug.edu.gh 37 speech problems and epilepsy (Birbeck et al., 2010; Brewster et al., 1990; Newton and Krishna, 1998). There is also evidence that suggests that some children who appear to have made a complete neurological recovery from cerebral malaria may develop significant cognitive problems (attention deficits, difficulty with planning and initiating tasks and language problems), which can adversely affect school performance and persist for years after the attack (Brewster et al., 1990; Idro et al., 2007; Njuguna and Newton, 2004). 2. 5.1 Pathogenesis of cerebral malaria There has been continuous effort to understand the pathogenesis of coma in paediatric CM because it is not clearly known what mechanisms determine the outcome of the illness. One major issue in the pathogenesis of paediatric CM is the nature of tissue injury that leads to severe central nervous system (CNS) damage and death in some of the infected children (Dorovini-Zis et al., 2011). Histopathological analysis of brain tissue from CM patients at autopsy has identified large numbers of P. falciparum iRBCs sequestered in the cerebral vessels with cerebral edema and localized haemorrhages (Aikawa et al., 1990; MacPherson et al., 1985; Pongponratn et al., 1991; Porta et al., 1992; Taylor et al., 2004a). In this regard two main hypothesis have been proposed for human cerebral malaria. The first being the mechanical hypothesis which involve impaired tissue perfusion because of sequestration of parasitized erythrocytes and immune-mediated injury secondary to host responses to parasite products. The second is an immune-pathological hypothesis which proposes that hyper-inflammatory responses responsible for eliminating P. falciparum parasite causes cerebral edema resulting from increased permeability and dysfunction of the blood- University of Ghana http://ugspace.ug.edu.gh 38 brain barrier (BBB) and organ failure (Brown et al., 1999; MacPherson et al., 1985). Figure 2.3 below attempts to summarize the mechanical hypothesis of the pathogenesis of human CM. Figure 2. 3 Diagram showing proposed mechanical hypothesis of the pathogenesis of cerebral malaria. Adapted from Chen et al. (2000) 2.5.2 Sequestration in CM It is now widely accepted that the histopathologic hallmark of CM is sequestration of infected red blood cells in the microcirculation of the brain and retina (Gyan et al., 2009). Approximately 16 hours after invasion of the red blood cells (RBCs), structural changes occur on the surface of these iRBCs and this results in the increase in their rigidity and adhesiveness to endothelial cells (Gardner et al., 1996). Owing to the increased adhesiveness, the red cells infected with late stages of P. falciparum (during the second half of the 48 hour life cycle) adhere to the capillary and University of Ghana http://ugspace.ug.edu.gh 39 postcapillary venular endothelium in the deep microvasculature. This is a process that allows the parasite to avoid splenic clearance mechanisms, but comes at a cost to the host. An important difference between P. falciparum and other human malarias is the way in which P. falciparum modifies the surface of the RBCs so that asexual parasites and gametocytes can adhere to the endothelium (Miller et al., 2002). This is facilitated by excessive inflammatory conditions that contribute to the adhesion of erythrocytes infected with P. falciparum to endothelial cells in brain capillaries (Schofield and Grau, 2005). Adherence to the microvasculature is promoted by the expression of parasite protein including Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of the infected red blood cell (Baruch et al., 1995; Rowe et al., 2009; Su et al., 1995). This protein is expressed by the parasite and exported to the surface of the infected erythrocyte, where it mediates adhesion to various host cell adhesion receptors (Baruch et al., 1995; Turner et al., 1994). The most well- described endothelium receptors for PfEMP1 are ICAM-1 in the brain, although binding to other receptors, including thrombospondin (TSP), PECAM-1, P-selectin, E-selectin, CD 36, CSA and VCAM has been reported (Gardner et al., 1996; Ockenhouse et al., 1992; Roberts et al., 1985; Rock et al., 1988). Sequestration of the growing P. falciparum parasites in these deeper tissues provides them the microaerophilic venous environment that is better suited for their maturation and the adhesion to endothelium allows them to escape clearance by the spleen and to hide from the immune system. The structural changes in parasitized RBCs results in the adherence of these parasitized RBCs to uninfected red cells leading to the formation of rosettes (Newbold et al., 1999) as shown in Figure 2.3 above. University of Ghana http://ugspace.ug.edu.gh 40 A detailed postmortem evaluation of cerebral microvessel sequestration in fatal pediatric CM has shown the presence of parasitized RBC sequestration in all patients with CM and an association of sequestration with microvascular pathology in 75% of patients with cerebral malaria (Taylor et al., 2004a). Figure 2. 4 Coronal section of the brain showing iRBC sequestration and microvascular thrombosis in fatal cerebral malaria. Adapted from Moxon et al. (2013) 2.5.3 Role of cytokines and immune mediators in CM The body’s response to the presence of infected RBC and subsequent disruption of microvascular blood flow as a result of sequestration may cause localized endothelial dysfunction. In addition University of Ghana http://ugspace.ug.edu.gh 41 induction of proinflammatory and pro-adhesive molecules may compromise the integrity of the endothelial barrier (Francischetti et al., 2008; Miller et al., 2013; Moxon et al., 2009). There is now overwhelming evidence that within cerebral vessels, adherent iRBCs induce endothelial activation, with several consequences (Dorovini-Zis et al., 2011). These consequences may be results of the host response to parasite products. Cytokines such as tumour necrosis factor (TNF)-α and interleukin (IL)-1, IL-6 and neuro-active mediators such as nitric oxide (NO) are produced as a result of sequestration of parasitized RBCs in the microvasculature (Figure 2.3). These cytokines are produced to inhibit parasite growth and promote parasite killing (Haidaris et al., 1983). These mediators can however, be toxic to the central nervous system (CNS) when overproduced. These mediators may also cause inflammations of the endothelium as morphological alterations of brain endothelium at the site of iRBC sequestration have been described (Pongponratn et al., 2003). Excessive levels of these cytokines have been associated with severe disease (Day et al., 1999). Children with CM have recorded significantly higher plasma levels of TNF-α than those with mild disease (Babikir, 2010). Malaria antigens have been shown to induce cytokine production. One major antigen is P. falciparum glycosylphosphatidylinositol (PfGPI), which are released from the iRBC when it ruptures at the schizont stage (Krishnegowda et al., 2005). PfGPI has also been shown to be involved in the production of purified TNF- α release from macrophages (Schofield and Hackett, 1993). TNF- α levels in peripheral blood and brain tissues have been shown to be elevated in patients with CM compared to those with uncomplicated malaria (Brown et al., 1999) and high University of Ghana http://ugspace.ug.edu.gh 42 levels in P. falciparum infection have been associated with increased soluble ICAM-1, a marker of endothelial activation (McGuire et al., 1996). Other cytokines such as IFN- gamma- produced as part of the early response to P. falciparum infection (Hensmann and Kwiatkowski, 2001) is also known to sensitize monocytes to produce increased levels of pro-inflammatory cytokines (such as TNF- α ) when exposed to parasite inflammatory mediators (Grau et al., 1989). Decreased levels of IL-10 and transforming growth factor beta (TGF-beta), which are major anti- inflammatory cytokines, has been associated with poor outcome in malaria (Day et al., 1999). Other studies have also shown high ratios of pro:anti-inflammatory cytokines (Perkins et al., 2000). The body’s inability to regulate the pro-inflammatory cascade, has been suggested to play a role in severe malaria disease including cerebral malaria. 2. 5.4 Endothelial activation and blood-brain barrier integrity in CM The brain contains a network of blood vessels which are necessary for providing nutrients and oxygen, and for removing carbon dioxide and waste (i.e. urea, creatinine, etc.). This network of capillaries together with the glia form a protective barrier called the blood-brain barrier (BBB). This barrier prevents large molecules and pathogens in the blood from entering the brain tissues and from altering the brain’s functions (Cardoso et al., 2010; Neuwelt et al., 2011). The brain is very sensitive to blood chemistry variations and its homeostasis is tightly regulated (Levin et al., 2011). University of Ghana http://ugspace.ug.edu.gh 43 Maintenance of homeostasis is principally due to the brain endothelial cells, which are on the luminal side of the blood-brain barrier and correspond to the actual barrier site. Brain endothelial cells differ from those found in other tissues in many ways. They are attached by tight junctions of high electrical resistance preventing intercellular passage of molecules, and do not contain small openings called slit pores that allow the diffusion of molecules (Rénia et al., 2012). Brain endothelial cells also have important functions in mediating and regulating the immune response in the nervous system (Miller, 1999). Endothelial cells, line the inner surface of blood cells and form a structural barrier between the blood and the rest of the body. During vasculogenesis (the formation of new blood vessels) and angiogenesis (the growth and remodelling of existing blood vessels), proteins produced by endothelial cells and their underlying mural cells are critical for the migration and apposition of endothelial cells to each other and their supporting cells. While endothelial cell activation directs key immune responses, both activation and apoptosis may lead to a loss of the endothelial barrier integrity resulting in vascular leak and dysfunction in target organs preferably the brain. Many studies have been performed to uncover the extent of BBB alterations and their relationship to CM pathogenic processes (Adams et al., 2002; Medana and Turner, 2006). Two families that have been extensively studied for their role in normal and pathologic angiogenesis are: vascular endothelial growth factor (VEGF) and its receptors Flt-1 (fms-like tyrosine kinase-1, (VEGFR-1) and Flk-1 (VEGFR-2); and angiopoietin-1 and angiopoietin-2 and their cognate receptor Tie-2 (Risau, 1997). Changes in these two proteins have considerable effect in endothelial activation and dysfunction. University of Ghana http://ugspace.ug.edu.gh 44 VEGF is a pro-vasculogenic, pro-angiogenic and pro-inflammatory protein that signals through two main receptors expressed on the endothelium, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1). VEGFR-2 appears to be the dominant signaling receptor necessary for inducing vascular permeability and vessel formation, whereas VEGFR-1 may serve to modulate VEGF signaling (Yancopoulos et al., 2000). VEGF is critical for vasculogenesis and angiogenesis and acts as a key destabilizing force during vessel remodelling. It has also been shown to influence other biomarkers such as angiopoietins that have been implicated in pathogenesis of cerebral malaria (Lobov et al., 2002). 2.5.5 Role of Angiopoietin 1 and 2 in CM The angiopoietins are among the most widely studied biomarkers involved in endothelial activation and dysfunction in diseases. They are important angiogenic proteins that are antagonistic ligands of the Tie-2 receptor, which belongs to a family of vascular tyrosine kinase receptors expressed primarily in endothelial cells (Page and Liles, 2013). These biomarkers have been identified as indicators of malarial disease severity (Fiedler and Augustin, 2006; Larkin et al., 2009). In healthy conditions, serum concentration of angiopoietin 1 (Ang-1) is higher than that of angiopoietin 2 (Ang-2). This favours the binding of Ang-1 to the Tie-2 receptor. Ang-1 is constitutively produced by pericytes and vascular smooth muscle cells that surround the endothelial cell and promote vascular stability and quiescence (Fiedler and Augustin, 2006). It is also expressed widely in normal adult tissues and is usually constitutively expressed in brain endothelium (Chittiboina et al., 2013). Ang-2 on the other hand is contained within Weibel-Palade University of Ghana http://ugspace.ug.edu.gh 45 bodies (WPB) in the endothelium and can be rapidly mobilized and released upon endothelial activation or exposure to inflammatory stimuli. The preferential binding of Ang-1 to the Tie-2 receptor in normal conditions therefore initiates pro-survival pathways and inhibit pro- inflammatory pathways. The net result is endothelial cell quiescence (Page and Liles, 2013). In contrast, inflammation stimulates Weibel–Palade body exocytosis and the release of Ang-2, allowing it to preferentially bind the Tie-2 receptor and promote proinflammatory and pro- thrombotic pathways, as well as microvascular leak or damage as shown in Figure 2.5 below. Ang 2 is also released alongside other WPB products such as von Willebrand factor (VWF) and its propeptide (Fiedler et al., 2004). Figure 2. 5 Proposed mechanism of the role of Angiopoietins in immunopathogenesis of CM. Adapted from Conroy (2012) University of Ghana http://ugspace.ug.edu.gh 46 In quiescence, clusters of Ang-1 aggregate and activate the transmembrane receptor tyrosine kinase, Tie-2, which is highly specifically expressed on endothelial cells. Tie-2 signals into the cell to favor phenotypes such as fortification of barrier function. In CM, Ang-2 is up regulated as a result of its release from the WBP and is believed to antagonize Ang-1. The tonic homeostatic signaling through Tie-2 is attenuated, contributing to the vascular leak and inflammation observed in CM. Ang-2 has also been shown to induce the expression of ICAM-1 and VCAM-1 at sub-saturating concentrations of TNF (Fiedler et al., 2006). The overexpression of Ang-2 destabilizes quiescent endothelial cells (EC) through an internal autocrine loop and leads to EC detachment and a vessel regression (Hu and Cheng, 2009). Elevated levels of Ang-2 have been observed to increase mortality in diseases such as sepsis (Kümpers et al., 2009; van der Heijden et al., 2009) and have been reported to be predictive of mortality in certain subsets of critically ill patients, including those requiring renal replacement therapy and in children with septic (Giuliano Jr et al., 2007) Ang-2 has also been shown to promote several activities in the microvasculature including the migration of endothelial cells in the presence of VEGF whiles in absence of VEGF, Ang-2 promotes endothelial cell death and vessel regression (Lobov et al., 2002). A change in the normally low Ang-2:Ang-1 ratio may give an indication of endothelial dysfunction and could be as a result of either a decrease in Ang-1 or an increase Ang-2, or both (Lukasz et al., 2008). The ratio of Ang-2/Ang-1 in serum therefore serves as a good predictor of disease severity University of Ghana http://ugspace.ug.edu.gh 47 thereby implicating them in various disease pathogenesis (Page and Liles, 2013). Decreased levels of Ang-1 and an up regulated Ang-2 has been associated with early phase injury in the endothelium of rats (Chittiboina et al., 2013). In Plasmodium falciparum malaria, plasma Ang-2 levels have been found to be higher in patients with severe than non-severe disease. However, Ang-1 has been identified as a more consistent diagnostic biomarker in malaria, discriminating between cerebral malaria and uncomplicated malaria. (Lovegrove et al., 2009). Furthermore, in a study in malaria-exposed pregnant women, Plasmodium falciparum infection was associated with a decrease in maternal plasma Ang-1 levels and an increase in the Ang-2: Ang-1 ratio. Ang-1 levels were recovered after treatment of peripheral parasitemia (Silver et al., 2010). Low birth weight have also been associated with increased Ang-2:Ang-1 ratios (Silver et al., 2010). 2.5.6 Role of thrombomodulin in CM Thrombomodulin (TM) is an integral membrane glycoprotein that has a major role in the regulation of intravascular coagulation (Sadler, 1997). TM is present in large quantities on the surface of the endothelium, particularly in the microcirculation, where it acts as an anticoagulant (Page and Liles, 2013) TM is expressed at a lower constitutive levels in the brain compared with other organs. This glycoprotein forms a complex with thrombin and this complex alters the function of thrombin, the main pro-coagulant enzyme, to an anticoagulant through activation of the protein C pathway University of Ghana http://ugspace.ug.edu.gh 48 (Salomaa et al., 1999) . The TM-thrombin complex also prevents thrombin form converting fibrinogen to fibrin and thereby preventing coagulation (Levi and Van Der Poll, 2013). TM is known to exhibit an anti-inflammatory effect, regulate cell adhesion and proliferation through its lectin-binding domain. (Kodama et al., 1990). Reduced expression of TM on the cell surface have been observed in activated endothelium and this indicates the shedding of the molecule into soluble forms, soluble TM (sTM) (Page and Liles, 2013). Studies in the early 90’s had stated that the plasma soluble TM is derived from the degradation of that on endothelial cell membrane by various proteases during the process of endothelial cell injury and is then released into circulation without any evidence of active secretion (Ishii et al., 1991). Using a mouse model, (Esmon, 2000) demonstrated that, increased serum TM level is associated with a decreased expression of TM on the endothelial surface in vivo. Nevertheless, elevated serum TM levels are found in diseases associated with locally increased levels of inflammatory cytokines such as in malaria, dengue fever, sepsis and other diseases and syndromes such as cardiovascular diseases, acute coronary syndrome, pulmonary thromboembolism, and severe hemorrhage (Salomaa et al., 1999). In malaria, soluble TM levels have been observed to be higher in severe conditions compared to uncomplicated conditions (Faust et al., 2001). In a study conducted by Mita-Mendoza et al. (2013) using Malian children with uncomplicated and non-cerebral severe malaria, they showed that sTM levels was elevated during infection and declined with convalescence. They also showed that the levels of sTM correlated with both parasitemia and disease severity, and were higher in children with severe malaria than in those with uncomplicated malaria. In another malaria study by Maya University of Ghana http://ugspace.ug.edu.gh 49 et al. (2008), sTM levels was higher in uncomplicated P. falciparum malaria than in uninfected healthy individuals, and the sTM levels positively correlated with levels of pro-inflammatory cytokines and anaemia which is a marker of disease severity. The elevated serum levels of TM in both studies suggests that endothelial cell dysfunction occurs in the P. falciparum malaria. sTM has been proposed as both a diagnostic and prognostic tool of endothelial activation or dysfunction (Faust et al., 2001). 2.5.7 Role of endothelial protein C receptor (EPCR) in CM EPCR is a type 1 transmembrane protein that is expressed primarily by endothelial cells of the large blood vessels (Laszik et al., 1997). It is homologous to major histocompatibility complex class I/CD1 family proteins, that is expressed mainly on the luminal surface of aortic endothelial cell, surface of monocytes, natural killer cells, neutrophils, eosinophils, immature hematopoietic stem cells, brain capillary endothelial cells and embryonic giant trophoblast (Stephenson et al., 2006). However it is expressed at lower constitutive levels in the brain compared with other organs (Moxon et al., 2013). EPCR is one of the most important components of the protein C (PC) pathway, classically known as the anticoagulant system. Studies have shown that mechanisms of the protein C anticoagulant pathway is triggered when thrombin binds to the endothelial cell receptor, TM (Castellino, 1995; Esmon et al., 1999) as has been stated earlier. This complex activates protein C to generate the anticoagulant enzyme activated protein C (APC), which, in complex with protein S, inhibits coagulation by inactivating two critical regulatory proteins, factors Va and VIIIa. This pathway plays a critical role in the negative regulation of blood coagulation, as evidenced by the fact that total deficiencies of protein C or protein S are associated with severe and life-threatening thrombotic complications (Esmon and Schwarz, 1995). University of Ghana http://ugspace.ug.edu.gh 50 EPCR also exists in plasma soluble form (sEPCR) that binds PC and APC with similar affinity (Fukudome et al., 1996; Regan et al., 1996). sEPCR may result from inflammation or activation of the endothelium and subsequent shedding of these receptor (Moxon et al., 2013). Loss of EC bound EPCR and increasing levels of soluble forms of EPCR have been demonstrated in a study of Malawian children with CM (Moxon et al., 2013). A recent study on children in Benin has also associated high plasma levels of sEPCR with increased mortality in children with CM (Moussiliou et al., 2014). This studies have suggest strongly that increased levels of this receptor in plasma results from a pathophysiological mechanism of CM and measurement of soluble EPCR at initial clinical evaluation could predict severe malaria. In a recent study by Turner et al, (2013), EPCR was observed to act as the endothelial receptor for PfEMP1 domain cassettes 8 and 13. The study as shown in Figure 2.6 demonstrated that parasites causing severe malaria exhibited stronger EPCR binding than parasites from children with uncomplicated malaria. Of considerable interest and importance, PfEMP1 was shown to bind EPCR near or at the same region of APC, implying that EPCR-mediated cytoadhesion likely inhibits APC-mediated EPCR-dependent cytoprotective effects on endothelial cells (Turner et al., 2013). University of Ghana http://ugspace.ug.edu.gh 51 Figure 2. 6 Role of EPCR in CM. Adapted from Turner et al. (2013) 2.6 HRP 2 and Cerebral Malaria Detection of malaria parasites on blood smears from peripheral blood and the presence of fever are the commonly used case definitions of malaria. However, in malaria-endemic settings asymptomatic parasitaemia complicates the diagnosis of malaria. The presence of parasites in peripheral blood, lacks specificity as symptoms of severe forms of malaria are nonspecific and can have different etiologies (English et al., 1996). Peripheral blood parasitaemia does not represent the sequestered parasite burden, which is pivotal to the pathophysiology of severe falciparum malaria (Hendriksen et al., 2013). Sequestration of asexual parasites occur in the second half of the erythrocytic stage of the life cycle and this prevents detection of these parasites in peripheral blood films (Silamut et al., 1999b). Autopsies of cerebral malaria have demonstrated 26–40 times the burden of Plasmodium falciparum parasites in the deep tissue circulation of the brain compared to peripheral blood (Pongponratn et al., 2003; Silamut et al., 1999b). This phenomenon may University of Ghana http://ugspace.ug.edu.gh 52 possibly explain the poor association between malaria severity and parasitemia measured by peripheral blood microscopy. Plasmodium falciparum Histidine-Rich Protein 2 (PfHRP2) is a parasite-derived water-soluble protein which is released in discrete amounts into the plasma, predominantly during schizont rupture (Desakorn et al., 2005). Released PfHRP2 is distributed over the plasma volume and, therefore, its concentration in plasma reflects the total body parasite burden, including the sequestered parasites. Studies involving Asian adults (Dondorp et al., 2005) and African children (Rubach et al., 2012) show that, in contrast with the peripheral blood parasite density, the plasma PfHRP2 concentration correlates strongly with disease severity and outcome. Other studies have shown that elevated plasma pfHRP2 concentrations can identify children with histologically confirmed cerebral malaria (Seydel et al., 2012) and can distinguish severe malaria from coincidental or uncomplicated malaria (Hendriksen et al., 2013). Higher plasma concentrations of PfHRP2 (>1700ng/mL) has been shown to be a more field-friendly approach to confirming the diagnosis of CM compared to other techniques such as postmortem sampling or ophthalmoscopy (Seydel et al., 2012). 2.7 Endothelial Cells (ECs) The endothelium plays a pivotal role in the regulation of several biological processes relevant to clinical investigators such as inflammation, homeostasis and angiogenesis (Cines et al., 1998). The endothelium is subjected to several pathophysiological stimuli including that from pro- inflammatory cytokines, growth factors, infectious agents, lipoproteins, and oxidative stress University of Ghana http://ugspace.ug.edu.gh 53 (Dignat‐George and Sampol, 2000). Prolonged subjection of the endothelium by these environmental pressures subsequently leads to dysfunction and damage (Dignat‐George and Sampol, 2000; Goon et al., 2006b). 2.7.1 Endothelial progenitor cell (EPC) and endothelial repair Endothelial repair may occur by migration and proliferation of surrounding mature endothelial cells. Replication of local endothelial cells has been shown to be insufficient in the repair of extensive endothelial damage (Asahara et al., 1999). It has been shown that, these mature endothelial cells are terminally differentiated cells with a low proliferative potential and their capacity to substitute damaged endothelial cells (Hristov et al., 2003a) . Evidence suggest the presence of endothelial precursors in peripheral blood and these precursors originate from the bone marrow (Asahara et al., 1997). These precursor cells have the potential to differentiate into mature endothelial cells and therefore they have been termed endothelial progenitor cells (Hristov and Weber, 2004). The presence of these bone marrow derived EPCs in the peripheral and umbilical cord blood had been demonstrated by (Asahara et al., 1999). Bone marrow derived endothelial progenitor cells are thus known to migrate to sites of endothelial damage to augment the local response by incorporation into the microvasculature (Lin et al., 2000). Interest in EPCs arises because of their potential as stem cells and thus providers of therapeutic neovascularization and the repair of existing, damaged endothelium (Blann and Pretorius, 2006). EPCs account for approximately 0.1% of peripheral blood, have proliferative potential, and can differentiate into mature circulating endothelial cells (Thomas et al., 2009). When required for vascular repair/angiogenesis or in cases of vascular stress, EPCs enter the peripheral blood and University of Ghana http://ugspace.ug.edu.gh 54 migrate to areas of endothelial damage, differentiate and begin the reparative process as shown in Figure 2.7 below (Real et al., 2008). Figure 2. 7 Circulating EPC contribute to endothelial repair (Hristov et al., 2003a) Circulating EPC levels can also provide clinical information on the atherosclerotic burden and even on the future cardiovascular risk (Fadini et al., 2012). Low EPC level has been demonstrated in patients with unstable angina and cerebrovascular (George et al., 2004). Transfusion of EPCs into animal models have been shown to reduce neo-intima formation after vascular injury (Werner et al., 2003) These considerable interests in exploiting the function of bone marrow derived EPC are thus being extended to severe malaria. A study by Gyan et al., (2009) determined EPC levels in Ghanaian children with cerebral and uncomplicated malaria as well as healthy children. The study associated University of Ghana http://ugspace.ug.edu.gh 55 decreased levels of EPC with cerebral malaria, thus placing cerebral malaria within the context of current paradigms of micro vascular repair. 2.7.2 Circulating endothelial cells (CEC) Endothelial cell damage or detachment usually results from continuous or exaggerated endothelial activation by environmental pressures (Figure 2.8). Figure 2. 8 Schematic representation of endothelial damage: adapted from Dignat‐George and Sampol (2000). Detached endothelial cells circulate in peripheral blood and are often termed circulating endothelial cells (CECs). Some CECs have phenotypes compatible with terminally differentiated endothelial cells in some cases being apoptotic or necrotic and thus most likely derived from the University of Ghana http://ugspace.ug.edu.gh 56 turnover of vessel walls. The presence of CECs has been recognized as a useful marker of vascular damage (Goon et al., 2006b). Usually absent in the blood of healthy individuals, CEC counts are elevated in diseases hallmarked by the presence of vascular insult, such as sickle cell anemia, acute myocardial infarction (Goon et al., 2006b). Other studies have also demonstrated increased CECs in other disorders encompassing vascular, autoimmune, infectious and ischemic diseases (Bertolini et al., 2006). Some cancer patients have also demonstrated increased CEC counts (Farace et al., 2007). CECs are considered ‘rare’ cells with a consensus around their level ranging from 0 to 1500 cells per millilitre of blood (Dignat‐ George and Sampol, 2000). In healthy subjects, a low basal level of endothelial turnover, respectively very low amounts of circulating and vessel wall–derived ECs (1 to 3/mL blood), has been described (Dignat‐George and Sampol, 2000). Increased numbers, often up to 10-fold or more, are found in a broad tranche of diseases and conditions associated with vascular perturbation or damage and broadly speaking, correlate with plasma and physiological markers of endothelial damage/dysfunction such as flow mediated dilatation, von Williebrand factor (vWf), and soluble E selectin (Makin et al., 2004b). Subpopulations of CECs have been shown to express E-selectin and other markers of activation (Bull et al., 2003), and also potentially bio-active tissue factor and thrombomodulin (Woywodt et al., 2003). In various disease condition, the longitudinal quantitation of CECs showed that their levels vary according to the clinical evolution. Levels in patients who are acutely ill are higher than those in patients in clinical remission or in recovery phases of the disease (Dignat‐George and Sampol, 2000). University of Ghana http://ugspace.ug.edu.gh 57 Plasmodium falciparum infection represents a major endothelial infection as a result of sequestration of infected RBC on the endothelium. However, very little is known of circulating endothelial cells in P. falciparum infection. Studies with balb/c mice infected with P. berghei endothelial cells were correlated with cerebral symptoms and death (Neill and Hunt, 1992). CECs therefore show promise as a prognostic marker for cerebral malaria. 2.7.3 CEC and EPC identification and quantification Immunomagnetic bead capture method and flow cytometry (FC) are the most common and widely accepted techniques of CEC and EPC enumeration. Immunomagnetic bead capture method which is a well validated technique is notably labour extensive and requires a high degree of operator skills (Woywodt et al., 2006). Flow cytometry on the hand offers a multi-marker approach to EPC and CEC estimation, involving the concurrent use of endothelial-associated cellular markers (Goon et al., 2006a). These cellular markers are expressed as surface receptors on the cells and are used to distinguish between CECs, EPCs and hematopoietic stem cells (HSCs) among others. Monoclonal antibodies against these surface receptors have made detection and quantification of these cells more practical. Circulating EPC have been identified and enumerated by flow cytometry by the expression of progenitor receptors, CD34 and CD133 and endothelial vascular endothelial growth factor receptor 2 (VEGFR2) also known as CD309 (Asahara et al., 1997). Earlier studies in the field have reported that CD34 positive (CD34+) and VEGFR2+ cells purified from various sources such as umbilical cord blood (UCB), peripheral blood (PB) and bone marrow are able to generate ECs in vitro, suggesting that CD34+ cells contains cEPCs (Asahara et al., 1997; Shi et al., 1998). Many University of Ghana http://ugspace.ug.edu.gh 58 investigators therefore define EPCs by means of the co-expression of CD34 and VEGFR2 (Bertolini et al., 2006; Shi et al., 1998). Others have used a combination of CD34 and a more immature marker, CD133 to select for putative EPCs (Gehling et al., 2000; Gyan et al., 2009). However, it has been reported that CD34+CD133+VEGFR2+ cells do not only give rise to endothelial cells but rather are a distinct subpopulation of HSCs (Timmermans et al., 2009; Yoder, 2009). Studies have indicated that about 90% of CD34+ progenitor cells express CD45 at low intensity (CD45dim), whereas less than 10% are CD45-negative (Fadini et al., 2012). Hence a true population of EPCs may be defined as CD45dim/CD34+/VEGFR-2+/CD133+ (Jain et al., 2012; Kondo et al., 2004). CEC estimation like EPCs involve the concurrent use of endothelial-associated cellular markers such as CD31, CD34, CD144, CD62E, CD105, CD106, CD146 and KDR, enabling better distinguish mature CECs from other cells such as lymphocytes and haematopoietic stem cells (Mancuso and Bertolini, 2010). CECs have been defined as negative for the leukocyte and HSC markers (CD45 and CD11b), positive for endothelial marker CD31 and CD34, negative for activation markers CD105 and CD106, and negative for the progenitor cell marker CD133 (Erdbruegger et al., 2006; Goon et al., 2006b). However, examination of transcripts of cell surface markers among endothelial cells have revealed the expression of CD133 by brain endothelial cells and partially by endothelial cells in the eye, testis and skin (Nolan et al., 2013). CECs are known to express CD31 at high or bright intensity and hence CD45-/CD34+/CD31bright/CD133- (Mund et al., 2012) or CD11b-/CD34+/CD31bright/CD133- may define putative CECs. For the purpose of CECs originating from brain endothelium, CECs may be defined as CD11b- /CD34+/CD31bright/CD133+. University of Ghana http://ugspace.ug.edu.gh 59 Table 2. 1 Definition of EPCs and CECs Markers Cells EPC CECs References CD 45 Pan-leukocyte marker, human hematopoietic stem cells (HSC) dim - (Duda et al., 2007) CD 11b Granulocytes,monocytes/macrophages , dendritic cells, NK cells, and subsets of T and B cells - - (Nolan et al., 2007) CD 31 Monocytes, platelets, granulocytes, endothelial cells and lymphocyte subsets dim Bright (Duda et al., 2007) CD 34 HSC and progenitors bone marrow stromal cells, capillary endothelial cells, embryonic fibroblasts, + + (Asahara et al., 1999; Lin et al., 2000) CD 133 human hematopoietic stem cells, stem cells, endothelial progenitor cells, glioblastomas, neuronal, + (-/+) (Nolan et al., 2007; Peichev et al., 2000) CD309 (VEGFR2) endothelial cells, embryonic tissues, and megakaryocytes + + (Duda et al., 2007; Nolan et al., 2007) (-): no expression on cells surface dim: reduced intensity of expression (+): expression on cell surface bright: strong intensity of expression (-/+): expressed on cells from certain organs and absent in others University of Ghana http://ugspace.ug.edu.gh 60 CHAPTER THREE 3 MATERIALS AND METHODS 3.1 Chemicals, Reagents and Equipment The sources and manufacturers of reagents, buffers, solutions and equipments used in the study are shown in Appendix 1. 3.2 Study Sites The study was done in collaboration with five hospitals of the Ghana Health Service; The Princess Marie Louise Children’s Hospital (PML), La General Hospital, Tema General Hospital, Ledzokuku Krowoh Municipal Assembly Hospital (LEKMA) and the Ridge Hospital. These hospitals are the main referral hospitals located within the Greater Accra Region of Ghana where all cases of cerebral malaria cases are managed (Figure 3.1). Healthy controls for the study were recruited from the Hayward Nursery School and Osu Home School in La community and LEKMA cluster of schools in Teshie. The hospitals and the schools are located within two hours’ drive from Noguchi Memorial Institute for Medical Research (NMIMR), where the analysis of samples was done. University of Ghana http://ugspace.ug.edu.gh 61 Figure 3. 1 Map of Greater Accra region showing the location of the study sites (red) University of Ghana http://ugspace.ug.edu.gh 62 3.3 Study Design In a longitudinal cohort study, children (2-12 years) who presented with coma to the emergency of any of the affiliated hospitals were screened by paediatricians for cerebral malaria whiles those presenting to the Outpatient Department (OPD) of the hospitals were screened for uncomplicated malaria. Uninfected healthy children of similar ages as malaria patients were recruited from community schools within the Accra municipality. Informed consent was obtained from parents or guardians of children who qualified for the study. Patients recruited at the hospitals were thus categorized into two main groups, cerebral malaria and uncomplicated malaria, with further subgroups as shown in Figure 3.2. Uninfected healthy controls also constituted one group. Recruitment of study participants was done between May, 2012 and August, 2015. Samples were obtained at initial clinical presentation (baseline) and at each of the time point as indicated on Figure 3.2. For children presenting with CM, whole blood samples were taken at baseline, at the time of a clinical event (recovery), and at 7 and 14 days post recovery. For children with UM, whole blood samples were obtained at baseline, 7 and 14 days post-baseline. For a UM patient that develops CM, samples were taken at baseline, at the time of conversion to CM, at the time of recovery from CM, 7 and 14 days post recovery. For healthy controls, at baseline, 7 and 14 days). All children who presented with malaria to the hospitals were treated per the routine clinical procedures. University of Ghana http://ugspace.ug.edu.gh 63 Figure 3. 2 Schematic presentation of the study design Levels of chemokine/protease (SDF and MMP9), endothelial mediators (Ang-1 and -2), anticoagulant receptors (TM and EPCR) and P. falciparum parasite protein, HRP2, were assessed at each of the time points and subgroups. The levels of these biomarkers were also correlated with transition in disease severity or recovery in patients with CM, UM and healthy controls. For a pilot study on the stability of EPCs and CECs, six male volunteers (30 – 50 years) in the Department of Immunology of NMIMR were recruited after informed consents were obtained. Volunteers had no clinical signs of illness and were not on medication for any medical condition. Blood samples were obtained at one time point and stored in both EDTA and C-C BCT over a seven day period. University of Ghana http://ugspace.ug.edu.gh 64 3.4 Ethical clearance Ethical approval for the study was obtained from the Institutional Review Board (IRB) of the Noguchi Memorial Institute for Medical Research (NMIMR). Approval from Ghana Health Service and Ghana Education Service was obtained. Study participants were enrolled only after informed consent was obtained from their parents and guardians following their understanding of the objectives of the study (see appendix 2A and 2B for consent forms). Participants could opt out of study at any time since participating was voluntary. 3.5 Inclusion/Exclusion Criteria 3.5.1 Specific inclusion criteria For clinical malaria, presentation at any of the affiliated hospitals with a history of fever within the previous 24/48 hours or current fever (axillary temperature ≥ 37.5OC) plus 5 or more P. falciparum parasites per HPF (approx. 2500 /µl) and no other obvious cause found for the fever was enough for recruitment into study. For cerebral malaria, recruitment into the study was based on a patient being unconscious, with a score of <3 on the Blantyre coma scale (BCS) and being in coma for at least 60 minutes. In addition, patient should have no record of recent severe head trauma or other causes of coma or neurological diseases including meningitis/encephalitis (as assessed by lumbar puncture). Recovery from CM was defined as regaining of full consciousness (BCS> 3). The patient may or may not have neurologic sequelae, which was assessed by study physicians and any deficits were recorded and monitored for the duration of the study. UM was defined as clinical malaria without any WHO criteria for severe malaria such as SMA or respiratory distress. Patients were monitored University of Ghana http://ugspace.ug.edu.gh 65 briefly in the outpatient clinic at the discretion of the study physician prior to returning home. Unlike CM, UM patients were not hospitalized. Healthy controls were defined as uninfected children assessed by blood films and without any history of malaria treatment two weeks prior to recruitment into study. 3.5.2 Specific exclusion criteria Participants were excluded from the study if parent, guardian or proxy was unable to give signed informed consent and/or unwilling to comply with requirements of the protocol. Also, evidence of concomitant infection at time of enrollment including meningitis/encephalitis and bacteremia was enough to exclude a participant. History of any underlying disease that compromised the diagnosis and outcome of the illness including HIV infection (assessed only by history) also excluded participants. Diseases/conditions known to alter levels of EPCs or induce microvascular damage such as recent severe bleeding, sickle cell disease or trait, evidence of bacterial or viral infection, history of diabetes mellitus, cardiovascular disease, hypercholesterolemia, surgery within 1 month, bone fracture within 3 months, major trauma within 1 month (e.g., car accident), blood transfusion within 3 months were also considered as exclusion criteria. Children with severe malaria anaemia that received blood transfusions were also excluded from the study. 3.6 Blood Sample Collection Per routine clinical procedures, two millilitres (2ml) of venous blood was collected from each patient into EDTA tubes by trained phlebotomists for complete blood count (CBC), blood culture, University of Ghana http://ugspace.ug.edu.gh 66 sickling test and blood smears for malaria parasite count. In addition 3ml of venous blood were collected into heparinized (1ml) and EDTA (1ml) tubes and blood culture bottles (1ml) for laboratory analysis and immunological assays. For cerebral malaria patients, cerebrospinal fluid (CSF) samples were obtained through lumbar puncture done by trained paediatricians. For healthy controls, a total of 3ml of venous blood were collected into heparinized tubes (1ml) and EDTA tubes (2ml) for laboratory and immunological assays. For the pilot study on the stability of EPCs and CECs, ten milliliters (10 ml) of venous blood was collected from each of the six volunteers using Butterfly needles (BD); 5 ml of the blood was collected into a 5 ml EDTA and the other 5 ml into a Cyto-Chex BCT tube. 3.7 Sample Processing Time and Storage Blood samples for routine clinical procedures were taken immediately to the hospital laboratories for evaluation, whiles those for the research study were transported in cold ice chests to the laboratories of the Immunology Department, NMIMR CSF samples were transported to either the Korle Bu Teaching Hospital or the MDS-Lancet laboratory in Accra, for analysis. At NMIMR, a 400ul aliquot of EDTA treated blood were used for flow cytometry and the rest were separated into RBCs and plasma for storage at -30oC. Heparinized blood was also processed by centrifugation and separated into RBCs and platelet-free plasma. This was done by initial centrifugation at 1000 x g for 15 minutes and separated plasma at 10000 x g for 10 minutes. Both RBCs and platelet free plasma were stored at -30oC for further analysis. University of Ghana http://ugspace.ug.edu.gh 67 Blood samples in EDTA and C-Chex BCT tube for the pilot study were stored at 4oC for 30 minutes before use in assays. Four hundred microliters (400ul) each of EDTA and C-C BCT treated blood were aliquoted for flow cytometry and the rest stored at 4oC. Flow cytometric analysis was repeated for both stored EDTA and C-C BCT treated blood at day 1 (24hrs), day 2 (48hrs) and day 7. 3.8 Laboratory Evaluations/Assays Laboratory investigations on samples obtained were carried out at both the Hospital laboratories affiliated to the study and at the Noguchi Memorial Institute for Medical Research. Investigations done at the Hospital laboratories were for routine diagnostic purposes which included blood smears for malaria parasite identification and estimation as well as complete blood counts. Results from the hospital laboratories were made available to the Clinicians and the study team. Blood culture results and parasite densities obtained on samples at the research laboratories were made available immediately to clinicians to assist in clinical care. For healthy controls, all laboratory investigation were done at NMIMR. 3.8.1 Parasitology Hospital laboratories prepared blood smears per the routine protocol for the diagnosis of malaria. Briefly, about 10ul of whole blood were obtained from a finger puncture onto a pre-cleaned microscope glass slide and spread uniformly in a circle of diameter 1-2cm (thick film). The smears were left to dry at room temperature and stained with 10% Giemsa for 15 minutes. Stained slides University of Ghana http://ugspace.ug.edu.gh 68 were dried and examined under a light microscope (at x100) with immersion oil. Results obtained from blood smears were semi-quantitative. At the research laboratories at NMIMR, thick and thin blood smears were prepared from EDTA treated blood for confirmation of parasitaemia and determination of parasite densities. Blood smears were prepared according to WHO protocol (WHO, 1988; WHO, 1991). Both thick and thin films were prepared on the same slide as shown in Figure 3.2 below. Figure 3. 3 Thick and thin blood film Briefly, for the thick smears, a drop of about 7μl of blood was placed at one end of a microscope glass slide and evenly spread in a circle to a diameter of about 1cm. For the thin blood film, about 4μl of blood was placed close to the middle of the microscope glass slide. A spreader slide was inclined at an angle of 45O on the drop of blood. The blood was made to spread along the entire width of the spreader slide and pushed forward rapidly and smoothly. The prepared blood films were air-dried thoroughly. The thin blood film was fixed in absolute methanol for species identification. Both films were then stained with freshly prepared 10% Giemsa solution (in Phosphate buffer) and left to stain for 15 minutes, washed and examined under light microscope with immersion oil. Parasite densities were estimated using the WHO guidelines (WHO, 2010). University of Ghana http://ugspace.ug.edu.gh 69 3.8.2 Haematology For malaria patients recruited into the study, complete blood counts were done at the hospital laborartory on initial presentation and subsequent reviews. A haemotological analyzer was used to measure: haemoglobin levels, platelets counts, total white blood cell (WBC) counts, total red blood cell (RBC) counts, mean corpuscular volume (MCV), haematocrit (HCT), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin content (MCHC). Sickling tests were done for all patients and healthy controls recruited for the study. A drop of blood was placed on a clean microscope slide. An equal volume of the 2% sodium metabiosulphite solution was added to the blood and mixed thoroughly. The mixture was covered gently with a cover slip to avoid trapping air bubbles and to give a low oxygen tension and read under a microscope. 3.8.3 Bacteraemia evaluation Evaluation of bacteremia in patients was done according to Cheesbrough (1984). Briefly, blood sample (1ml) was obtained aseptically form each patient and added to 50ml broth medium. To reduce the risk of contamination, blood was collected from a peripheral vein by a qualified phlebotomist. Culture medium was kept in an incubator at 37oC and observed daily for seven days for any signs of haemolysis, production of gas, coagulation of the broth and turbidity above the red cell layer. If there was a sign of bacterial growth, a subculture was done on solid media and examined after 24 hours for bacteria growth. However, if there was no sign of bacterial growth, the culture was examined daily for seven days. University of Ghana http://ugspace.ug.edu.gh 70 Identification of bacteria growth was done by initially doing a Gram stain to differentiate Gram- positive from Gram-negative bacteria. 3.8.4 Flow cytometric analysis Flow cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light (Biosciences, 2000). This technique was used to evaluate the levels of EPCs and CECs in whole blood from the different groups of study participants Using a FACS Calibur flow cytometer (Figure 3.4). Figure 3. 4 Flow cytometer (FACS Calibur) 3.8.4.1 Processing of whole blood samples for flow cytometry EDTA treated whole blood samples were used for flow cytometry by following a routine in-house protocol at the Immunology Department, NMIMR. Briefly, panels of six 5ml polystyrene Falcon University of Ghana http://ugspace.ug.edu.gh 71 tubes (BD) were labeled (1-6) with permanent marker and different volumes of whole blood and Fc receptor blocking agent (IgG chrome) were added as indicated in Table 3.1 below. Antibodies against the various endothelial receptors considered in the study and their isotypes (positive and negative controls) were also added to the respective panels. Table 3. 1 Monoclonal antibodies against endothelial receptors Monoclonal antibodies and Fluorochromes Tube Whole Blood (ml) FITC (3μl) PE (3μl ) PerCP (3μl) APC (3μl) 1 50 Unstained 2 50 M-IgG1 k M-IgG1 M-IgG1k M-IgG 3 50 R-IgG 2b M-IgG1 M-IgG1k M-IgG 4 50 CD 15 CD 14 CD 4 CD 8 5 100 CD 11b CD 133 CD 34 CD 31 6 100 CD 45 CD 133 CD 34 CD 309(KDR) University of Ghana http://ugspace.ug.edu.gh 72 3.8.5 Quantification of cEPC and CEC in whole blood Circulating EPCs levels were determined by surface staining with receptor-specific fluorescent- labeled antibodies and expressed as percentage of CD45dim/VEGFR2+/CD34+/CD133+ cells in total leukocyte gate (G1) as shown in Figure 3.5. Figure 3. 5 cEPCs gating strategy and estimation Initial gating on all three major populations of WBCs (Lymphocytes, monocytes and granulocytes) [G1]. Gating on CD45dim population from initial gate G1, to exclude cells such as haematopoietic stem cells and leukocytes (G2). CD309 (VEGFR2) population within CD45dim population were gated (G3). Final gating of CD34+ and CD133+ population from G3 to enumerate cEPCs as CD45dim/VEGFR2/CD34+/CD133+. Forward Scatter S id e S ca tt er F o rw a rd S ca tt er CD45 VEGFR-2 F o rw a rd S ca tt er CD34 C D 1 3 3 G1 G2 G3 University of Ghana http://ugspace.ug.edu.gh 73 CEC levels were determined by surface staining with receptor-specific fluorescent-labeled antibodies and expressed as percentage of CD11b-/CD34+/CD31bright/CD133+ cells in total leukocyte gate (G1) as shown in Figure 3.6. Figure 3. 6 CEC gating strategy and estimation. Initial gating on all three major populations of WBCs [Lymphocytes, monocytes and granulocytes (LMN)] (G1). Gating on CD11b- and CD34+ population from initial gate G1. CD31bright and CD133+ population within G2 population were gated to enumerate CECs as CD11b- /CD34+/CD31bright/CD133+. CECs were estimated as percentage of CD11b- /CD34+/CD31bright/CD133+ cells of initial LMN gate G1. Forward Scatter S id e S c a tt e r CD11b C D 3 4 C D 1 3 3 CD31 G2 G1 University of Ghana http://ugspace.ug.edu.gh 74 3.8.6 Evaluation of chemokine/protease and endothelial biomarkers Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of SDF-1, MMP9, Ang-1 and Ang-2, soluble TM and soluble EPCR as well as parasite protein HRP2. Measurement of the endothelial biomarkers, Angiopoietin-1 and -2, soluble TM and soluble EPCR were obtained as duo sets from R&D. Coating of plates were done according to the manufacturer’s instructions. Ninety six (96)-well polystyrene microtitre plates were coated for each biomarker with 100μl per well of mouse anti-human antibody (capture antigen) specific for the biomarker and incubated overnight at room temperature. The plates were aspirated and washed using 400μl of wash buffer [0.05% Tween 20 in Phosphate Buffered Saline (PBS)] and blotted against clean paper towel after three washes to adequately remove excess unbound capture antigen. The plates were then blocked with 300μl per well of reagent diluent [1% Bovine Serum Albumin (BSA) in PBS] and incubated for an hour at room temperature. The plates were then aspirated and washed again. Hundred microlitres (100μl) of plasma samples (20 times diluted in PBS) were added per well for each of the plates coated for the different biomarkers. The plates were covered with an adhesive strip and incubated for 2 hours at room temperature on a horizontal orbital microplate shaker (0.12" orbit) set at 500 +/- 50 rpm. The wells were aspirated and washed three times with 400μl of wash buffer using a manifold dispenser. The plates were blotted with clean paper towels at the end of the wash to completely remove any liquid. Hundred microlitres (100μl) of detection antibody diluted in reagent diluent with normal goat serum (NGS) for each biomarker were added to the wells. The plates were covered with a different adhesive strip and incubated for 2 hours at room University of Ghana http://ugspace.ug.edu.gh 75 temperature on a shaker after which the plates were aspirated and washed three times as described above. Hundred microlitres (100μl) of working dilution of Streptavidin-HRP was added to each of the wells plates. The plates were covered with an adhesive strip and incubated for 20 minutes in the dark at room temperature. The plates were again aspirated and washed three times as described above. Hundred microlitres (100μl) of substrate solution for each biomarker was added to each corresponding well for colour development. The plates were then incubated for 20 minutes at room temperature in the dark. Stop solution (50μl) was added to each well to halt the colour change. The optical density was determined within 30 minutes, using the EL808 BioTek microplate reader set at 450 nm. The levels of each of the endothelial biomarkers were then determined from the optical densities. 3.8.7 Statistical Analysis Data generated in this study were mostly analyzed by both parametric and non-parametric tests. Kruskal Wallis test and Friedman test were used to determine differences between more than two groups. Dunn’s multiple comparison test was employed for pairwise comparison of groups where applicable. A t-test was employed when comparison involved only two groups. Characteristics of study participants were recorded as median with minimum and maximum values. Levels of marker considered in the thesis were presented as mean with measure of dispersion. P-values of less than 0.05 (two sided) were regarded as statistically significant. Statistical analyses and data presentation were done using GraphPad Prism (San Diego, CA). University of Ghana http://ugspace.ug.edu.gh 76 CHAPTER FOUR 4 RESULTS 4.1 Background and Demographics of Study Participants One hundred and fifty three (153) children between one and 12 years were recruited for the study. A total of 21 (13.7%) cases were disqualified based on the exclusion criteria in section 3.4.2. The total number of qualified cases were 132 consisting of 50 (38%) confirmed CM cases, 45 (34%) UM cases and 37 (28%) HC. Table 4.1 shows characteristics of qualified cases recruited for the study. A total of 8 CM resulted in fatalities. No deaths were recorded in UM cases over the four years of the study and none of the cases recruited as UM converted to CM throughout the study. Median age of healthy controls was higher compared to both CM and UM (P<0.0007, Kruskal Wallis and Dunn’s multiple comparison). There was no significant difference between the median ages of CM and UM cases (p>0.999). Hb levels were significantly lowest amongst the study groups (p<0.0001, Dunn’s Multiple comparison test). CM recorded the highest WBC counts compared to both UM and HC (p<0.0001, Dunn’s Multiple comparison test). No significant difference in the median WBC counts in UM and HC (p>0.999) was observed. RBC count in CM was significantly lower than both UM and HC (CM vs UM, p=0.0012: CM vs HC, p<0.0001, Kruskal wallis and Dunn’s multiple comparison). No statistical difference was observed between RBC levels in UM and CM. Kruskal Wallis and Dunn’s multiple comparison test shows significant differences (p<0.0001) in the HCT levels in the study groups, with CM recording the lowest (26.20%) and HC recording the highest (34.25%). University of Ghana http://ugspace.ug.edu.gh 77 Platelet count in HC was significantly higher than UM and CM (p<0.0001). No statistical significance was observed between the platelet count of UM and CM cases (p=0.326, Dunn’s Multiple comparison). A significant difference was observed between the median parasite densities of the cerebral malaria (840 parasites/uL/blood) and uncomplicated malaria (27,900 parasites/uL/blood) groups (P= 0.0023, t-test). There were significantly more parasite seen in the peripheral blood of the UM than in the CM. University of Ghana http://ugspace.ug.edu.gh 78 Table 4. 1 General Characteristics of study participants Characteristics Study Groups P value Cerebral Malaria Uncomplicated Malaria Healthy Control N 50 (38%) 45 (34%) 37 (28%) Age (years) 5.3 (1-12) 5.0 (1.5-11) 9 (1-12) * 0.0007 Sex (% male) 62.75∞ 56.82 36.36 <0.03 Hb (g/dL) 8.6 (5.9-14.90)β 10.75 (6-14.10) 12.00 (9.10 -14.20) <0.0001 WBC (103/uL) 10.65 (2.8 – 33.0) 6.70 (2.4 – 29.5) 6.450 (4.1 – 10.6) <0.0001 RBC (106/uL) 3.44 (2.30 – 6.04) 4.30 (2.62 – 5.52) 4.58 (3.5 – 5.66) <0.0001 HCT (%) 26.20 (18.90 – 43.10) 30.20 (19.00 – 40.20) 34.25 (25.00 – 41.20) <0.0001 Platelets (103/uL) 60.0 (20.0 – 550.0) 105.5 (21.0 – 400.0) 329.5 (209.0 – 573.0) § <0.0001 Parasite density (parasite/µl) 840 (1-304000)α 27900(1680-154000) - 0.0023 (t-test) Data represents the median and minimum and maximum values. P value was obtained by Kruskal-Wallis on Ranks. Post-hoc test was done by Dunn’s Multiple Test to detect significant differences between paired groups. T-test was used to compare parasite densities of CM and UM. * indicates that the median age of HC was higher than that of CM and UM. ∞ indicates there were significantly more males in CM than in UM and HC. β Indicates the Hb level in the CM group was significantly lower than UM and HC group (P < 0.001) following the Post-Hoc test. § indicates that median platelet count in HC group were significantly higher than UM and CM (P<0.05). α median parasite density in CM was lower than that of UM (p=0.0023, t-test) University of Ghana http://ugspace.ug.edu.gh 79 4.2 Stability of cells in C-C BCT 4.2.1 Stability of leukocyte populations in C-C BCT preservative Whole blood from healthy volunteers were stored in EDTA anticoagulant and C-C BCT blood preservative at 4oC and processed for flow cytometry on days 0 (30 minutes), 2 (48 hours) and 7. There was an overall discrimination of leukocyte populations in C-C BCT compared with that in EDTA-treated peripheral blood stored at 4oC (Figure 4.1). This is evident on day 7 in blood stored in C-C BCT where the three major leukocyte populations could be clearly gated or differentiated. Figure 4. 1 Leukocyte populations in whole blood preserved in C-C BCT preservative and EDTA anticoagulant over a seven day period. 4.2.2 Stability of EPCs in C-C BCT preservative EPC levels were estimated in adult whole blood preserved in C-C BCT at 4oC over a seven day period. Levels of EPC in C-C BCT at different time points were compared with that in EDTA University of Ghana http://ugspace.ug.edu.gh 80 anticoagulant at baseline (day 0). Mean cEPC levels were not significantly different between baseline EDTA samples (0.017%, 95% confidence interval 0.006- 0.028) and C-C BCT samples stored at 4oC for day 0 (0.038%, 95% CI 0.005- 0.072), day 1 (0.030%, 95% CI 0.017- 0.042) and day 2 (0.047%, 95% CI 0.019- 0.075) following pairwise comparisons, but the mean baseline EDTA blood cEPC levels were significantly lower (p=0.004) than that in whole blood preserved in C-C BCT for 7 days (0.070%, 95% CI 0.030- 0.10) [Figure 4.2]. T im e c o u rs e % c E P C E D T A D a y 0 C - C D A Y 0 C - C D a y 1 C - C D a y 2 C - C D a y 7 0 .0 0 0 .0 5 0 .1 0 0 .1 5 ** Figure 4. 2 Percentage of cEPC at different time points in whole blood preserved in C-C BCT. ** indicates lower cEPC levels in EDTA compared with C-C BCT day 7 ( p<0.01). 4.2.3 Stability of CECs in C-C BCT preservative CEC levels were estimated in adult whole blood preserved in C-C BCT at 4oC over a seven day period. Levels of CEC in C-C BCT at different time points were compared with that in EDTA University of Ghana http://ugspace.ug.edu.gh 81 anticoagulant at baseline (Figure 4.3). Mean CEC levels were not significantly different between baseline EDTA samples (0.0026%, 95% CI 0.001- 0.004) and C-C BCT samples stored at 4oC for day 0 (0.0012%, 95% CI 0.00008- 0.003), day 1 (0.002%, 95% CI 0.0001- 0.0035) and day 2 (0.003%, 95% CI 0.002- 0.005) following pairwise comparisons, but the mean baseline EDTA blood CEC levels were significantly higher (p=0.043) than that in whole blood preserved in C-C BCT for 7 days (0.0004%, 95% CI -0.0006- 0.001): T im e c o u rs e % C E C E D T A D a y 0 C - C D A Y 0 C - C D a y 1 C - C D a y 2 C - C D a y 7 -0 .0 0 2 0 .0 0 0 0 .0 0 2 0 .0 0 4 0 .0 0 6 * Figure 4. 3 Percentage frequency of CEC at different time points in whole blood preserved EDTA anticoagulant and in C-C BCT. ** indicates higher CEC levels in EDTA compared with C-C BCT day 7 (p<0.05). 4.2.4 Stability of common immune markers in C-C BCT Levels of common immune markers (CD4, CD8, CD14 and CD15) were determined in blood preserved in C-C BCT at 4oC over a seven day period. Levels of these markers at different time points were compared with that in EDTA anticoagulant at baseline as shown in Figure 4.4. No University of Ghana http://ugspace.ug.edu.gh 82 significant differences were observed between expression levels of CD4, CD8, CD15 over the seven day storage in C-C BCT when compared to EDTA (p>0.05, Friedman test and Dunn’s multiple comparison) in all cases. However, mean CD14 levels were significantly higher (**p=0.023 Dunn’s multiple comparison test) on day 7 (13.12%, 95% CI 7.123- 19.11) when compared with EDTA day 0 (4.67%, 95% CI 3.327- 6.016). Figure 4. 4 Percentage frequencies of some immune marker at different time points in whole blood preserved in C-C BCT. ** indicates higher CD14 levels compared to EDTA day 0 and first 2 days in C-C BCT. 4.2.5 Stability of endothelial and other markers in C-C BCT Levels of some endothelial and other markers (CD31, CD133, CD34, CD309, CD11b and CD45) were determined in whole blood preserved in C-C BCT at 4oC over a seven day period. Levels of University of Ghana http://ugspace.ug.edu.gh 83 these markers at different time points were compared with that in EDTA anticoagulant at baseline as shown in Figure 4.5. Percentage frequency of cells expressing CD133 were significantly higher (**p= 0.041) on day 7 in the preservative (18.98%, 95% CI 14.14 – 23.83) compared to baseline frequencies in EDTA (3.43%, 95% CI 1.66 – 5.183). CD309 levels on Day 7 (2.89%, 95% CI 1.834 – 3.95) in C-C BCT were significantly higher (*p=0.005, Dunn’s multiple comparison) compared to baseline frequencies in EDTA (0.76%, 95% CI 0.66 – 2.17). Percentage frequency of CD11b cells were significantly reduced (p=0.02, Dunn’s multiple comparison test) on day 2 in C- C BCT (8.032%, 95% CI 5.45 – 10.62) compared to baseline EDTA (20.26% 95% CI 12.13 – 28.39). CD45 frequencies were also significantly reduced (p=0.01, Dunn’s multiple comparison test) on day 7 in C-C BCT (95.07%, 95% CI 92.78 – 97.36) compared with EDTA day 0 (98.13%, 95% CI 97.42 – 98.83). Percentage frequencies of cells expression CD31 and CD34 were significantly stable in C-C BCT over the seven day period and levels were comparable with EDTA day 0. University of Ghana http://ugspace.ug.edu.gh 84 C D 3 0 9 (V E G F R -2 ) E D T A d a y 0 c - c d a y 0 c - c d a y 1 c - c d a y 2 c - c d a y 7 0 1 2 3 4 5 * C D 3 1 E D T A d a y 0 c - c d a y 0 c - c d a y 1 c - c d a y 2 c - c d a y 7 0 2 0 4 0 6 0 8 0 C D 1 3 3 E D T A d a y 0 c - c d a y 0 c - c d a y 1 c - c d a y 2 c - c d a y 7 0 5 1 0 1 5 2 0 2 5 ** C D 3 4 E D T A d a y 0 c - c d a y 0 c - c d a y 1 c - c d a y 2 c - c d a y 7 0 2 4 6 8 1 0 C D 1 1 b E D T A d a y 0 c - c d a y 0 c - c d a y 1 c - c d a y 2 c - c d a y 7 0 1 0 2 0 3 0 4 0 ** C D 4 5 E D T A d a y 0 c - c d a y 0 c - c d a y 1 c - c d a y 2 c - c d a y 7 8 5 9 0 9 5 1 0 0 1 0 5 *** Figure 4. 5 Percentage cell count of individual cell expressing EPC, CEC, haematopoietic and leukocyte receptors in whole blood preserved in Cyto-chex BCT at 4oC for seven days. Time Course % F re q u e n ci es ***p<0.0001 ** p<0.001 * p<0.05 University of Ghana http://ugspace.ug.edu.gh 85 4.3 Levels of cEPCs and CECs in the Study Groups 4.3.1 cEPC levels in different groups at baseline cEPC levels were estimated using the gating strategy in Figure 3.5. Comparing the baseline cEPC levels in the three study groups, mean cEPC levels in HC (0.083%) were significantly higher than levels in CM (0.042%, p=0.0071, Dunn’s multiple comparison test) but similar to levels in UM (0.140%, p=0.1414). Mean levels in UM were also significantly higher than those in CM (p<0.0001). C M U M H C 0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 *** ** G ro u p s % E P C Figure 4. 6 cEPCs levels at initial evaluation in the study groups. ***p<0.0001 ** p<0.001 University of Ghana http://ugspace.ug.edu.gh 86 4.3.2 Time course estimation of cEPC levels in different groups cEPC levels were determined during the time course of recovery from malaria infection (CM and UM) as well as in healthy children (Figure 4.7). cEPC levels were elevated during the time course of recovery from CM (p<0.0001), peaking at day seven post-recovery indicating repair or replacement of damaged endothelial cells in CM patients. Mean cEPC levels in CM: Day 0= 0.042% [95% confidence interval (CI) 0.034- 0.049], Recovery = 0.117% [95% CI -0.063- 0.171], Day 7= 0.157% [95% CI 0.076-0.238], Day 14 = 0.093% [95% CI 0.059-0.127]. No significant differences were observed in the pairwise comparison within the other study groups [UM (Day 0, day 7 and Day 14) p= 0.4226, HC (Day 0, day 7 and Day 14) p>0.9999)]. Data are presented as mean 95% confidence interval. D a y 0 R e c o v e r y D a y 7 D a y 1 4 D a y 0 D a y 7 D a y 1 4 D a y 0 D a y 7 D a y 1 4 0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 G ro u p s % E P C C M U M H C *** Figure 4. 7 Time course cEPC levels in study the groups. ***p<0.0001 University of Ghana http://ugspace.ug.edu.gh 87 4.3.3 CEC levels in different groups at initial presentation Using Lyse-wash protocol, CECs were quantified as CD11b-, CD34+, CD31bright and CD133+ using flow cytometry gating strategy shown in Figure 3.6. CM showed the highest CEC levels compared to UM or HC (Dunn's multiple comparisons test: CM vs UM **p=0.0037, CM vs HC *p=0.0413, Figure 4.8) suggesting significant endothelial damage in the CM patients. No significant difference was found between uncomplicated and healthy controls (p>0.9999). C M U M H C 0 .0 0 0 0 .0 0 5 0 .0 1 0 0 .0 1 5 0 .0 2 0 ** * G ro u p s % C E C Figure 4. 8 Percentage CECs at initial evaluation in the study groups ** p<0.001 * p<0.05 University of Ghana http://ugspace.ug.edu.gh 88 4.3.4 Time course evaluation of CECs CEC levels were determined during the time course of recovery from malaria infection (CM and UM) as well as in healthy children. Higher levels of CECs were observed in CM patients even at fourteen days post recovery from coma (Figure 4.9). UM patients showed a sharp increase and decrease in CEC levels day seven (p<0.001) and fourteen (p=0.002) post initial clinical evaluation respectively. Mean CEC levels in CM; Day 0= 0.003% (95% CI 0.002- 0.004), Recovery = 0.004% (95% CI -0.003- 0.006), Day 7= 0.006% (95% CI 0.003-0.008), Day 14 = 0.006% (95% CI 0.005-0.009). Mean CEC levels in UM; Day 0= 0.001% (95% confidence interval (CI) 0.0004- 0.002), Day 7= 0.009% (95% CI 0.006-0.011), Day 14 = 0.002% (95% CI 0.0009-0.003). G ro u p s % C E C D a y 0 R e c o v e r y D a y 7 D a y 1 4 D a y 0 D a y 7 D a y 1 4 D a y 0 D a y 7 D a y 1 4 0 .0 0 0 .0 1 0 .0 2 0 .0 3 C M U M H C * * * * * ** Figure 4. 9 Time course CEC levels in the study groups ***p<0.0001 ** p<0.001 University of Ghana http://ugspace.ug.edu.gh 89 4.4 Plasma levels of Ang-1 and Ang-2 Ang-1 and-2 levels were determined during the time course of recovery from malaria infection (CM and UM) as well as in healthy children (Table 4.2). Levels of Ang-2 in the study groups at initial evaluation did not show any statistically significant differences (p=0.124, Kruskal Wallis) even though CM patients had higher Ang-2 levels (5260pg/ml) compared to that in UM patients (4041pg/ml) and HC (4909pg/ml) suggesting microvascular leak or damage in CM patients. Ang- 2 levels did not also show any significant difference (p=0.147) in course of recovery from CM. However, UM showed significant increase (p=0.016) on Day 14 after initial clinical presentation. Ang-1 levels were significantly lower (p<0.05) in UM compared to both CM and healthy controls at initial presentation. Plasma levels of Ang-1 reduced (p=0.04) during coma but showed a sharp increase (p<0.001) seven days after recovery from coma indicating promotion of pro-survival pathways and endothelial cell quiescence. UM also showed a sharp increase (p=0.005) in Ang-1 level on day seven after initial presentation of malaria and maintained level on day 14. The ratio of Ang2:Ang1 showed a significant increase (p<0.05) at recovery and a subsequent decrease at days 7 in CM patient suggesting endothelial injury and restoration of endothelial integrity respectively. The ratio of these two endothelial mediators could not differentiate the state of the endothelium as no significant difference was observed between the Ang1:Ang2 ratios of CM, UM and HC (p>0.5, Kruskal Wallis). University of Ghana http://ugspace.ug.edu.gh 90 Table 4. 2 Time course Plasma levels of Ang-1 and -2 in the study groups Groups Biomarker Day 0 Recovery Day 7 Day 14 P (Kruskal Wallis) CM ANG2 (pg/ml) 5260 (4310-6120) 6287 (5275-7299) 4923 (4262-5585) 6021 (4846-7197) 0.147 ANG1 (pg/ml) 16072 (8215-23928) 8967# (4994-12940) 20609 (15470-25748) 18720 (12968-24471) ***<0.0001 ANG2:ANG1 (pg/ml) 0.493 (0.32-0.67) 0.895β (0.744-1.05) 0.287 (0.16-0.40) 0.408 (0.29-0.53) ***<0.0001 UM ANG2 (pg/ml) 4041α (3265-4818) 5354 (4285-6423) 5571 (4861-6280) **0.017 ANG1 (pg/ml) 9248∞ (6020-12475) 16451 (11496-21407) 15187 (11468-18906) **0.005 ANG2:ANG1 (pg/ml) 0.437 (0.29-0.59) 0.355 (0.24-0.47) 0.453 (0.34-0.57) 0.4026 HC ANG2 (pg/ml) 4909 (3048-6936) ANG1 (pg/ml) 20675 (17258-24092) ANG2:ANG1 0.318 (0.26-0.38) *** p<0.0001 ** p<0.05 #: Levels of Ang-1 is significantly lower (p<0.05) compared to all other time points in CM β: Ang-2:Ang-1 ratio significantly higher (p<0.05) compared to all time points in CM α: Significant lower levels of Ang-2 at day 0 compared to day 14 in UM patients ∞: Significant lower Ang-1 levels at day 0 compared with other time points in UM University of Ghana http://ugspace.ug.edu.gh 91 4.5 Levels of Endothelial Receptors 4.5.1 Baseline levels of soluble EPCR in the study groups Soluble EPCR levels were measured by ELISA at initial evaluation in the study groups (Figure 4.10). Baseline plasma levels of EPCR in the study groups showed CM patients with the highest levels (20.14pg/ml) compared to that of UM patients (16.82pg/ml) and HC (15.39pg/ml) (p<0.05, Kruskal Wallis). Post-hoc comparison of soluble EPCR levels in CM and UM did not reach statistical significance (p>0.05, Dunn’s multiple comparison test) even though CM showed higher levels. However, levels in CM was significantly higher compared with HC (p=0.002, Dunn’s multiple comparison test). C M U M H C 0 1 0 2 0 3 0 4 0 5 0 6 0 S tu d y g ro u p s E P C R l e v e l s ( p g / m l ) ** Figure 4. 10 Soluble EPCR levels in study the groups ** p<0.001 University of Ghana http://ugspace.ug.edu.gh 92 4.5.2 Time course levels of soluble EPCR in the study groups Plasma levels of soluble EPCR were determined at different time points in the recovery from malaria and in healthy controls (Figure 4.11). Time course levels did not reach statistical significance in the various study groups (CM, p= 0.31 and UM, p=0.29, Kruskal Wallis). G ro u p s E P C R l e v e l s ( p g / m l ) D a y 0 R E C D a y 7 D a y 1 4 D a y 0 D a y 7 D a y 1 4 H C 0 5 1 0 1 5 2 0 2 5 3 0 CM UM Figure 4. 11 Levels of soluble EPCR at different time points in the study groups 4.5.3 Baseline levels of soluble TM in the study groups Initial determination of soluble TM was done in the various study groups (Figure 4.12). Levels in CM was significantly higher (8084pg/ml) compared with UM (5785pg/ml) and HC (5076pg/ml) University of Ghana http://ugspace.ug.edu.gh 93 (CM vs UM, p=0.01: CM vs HC, p<0.0001, Kruskal Wallis and Dunn's multiple comparisons). Levels in HC and UM were not significantly different (p=0.16). C M U M H C 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 G ro u p s T M l e v e l s ( p g / m l ) * ** Figure 4. 12 Soluble TM levels in the study groups ** p<0.001 * p<0.05 University of Ghana http://ugspace.ug.edu.gh 94 4.5.4 Time course levels of soluble TM in the study groups Follow up plasma levels of soluble TM were determined in CM and UM patients (Figure 4.13). TM levels in CM significantly dropped (p<0.05, Kruskal Wallis) after initial clinical presentation (Day 0) compared to subsequent time points (Day 0 vs Rec, p=0.002, Day 0 vs Day 7, p=0.033, Day 0 vs Day 14, p=0.0001, Dunn's multiple comparisons). No significant difference was observed between recovery from coma and fourteen days after that. Similar significant drop in sTM levels was observed in the time course in UM resolution. (Day 0 vs Day 7: p=0.0013. Day 0 vs Day 14; p=0.01). G ro u p s T M l e v e l s ( p g / m l ) D a y 0 R e c D a y 7 D a y 1 4 D a y 0 D a y 7 D a y 1 4 H C 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 CM UM H C ** ** Figure 4. 13 Levels of soluble TM at different time points in the study groups ** p<0.001 University of Ghana http://ugspace.ug.edu.gh 95 4.6 HRP2 levels 4.6.1 Baseline levels of HRP 2 in the study groups HRP2 levels were determined in plasma of children in the various study group at baseline (Figure 4.14). CM showed the highest HRP2 levels (2281ng/ml) [p<0.0001, Kruskal Wallis] compared with both UM (353ng/ml) and HC (15ng/ml) (CM vs UM, ***p<0.0001: CM vs HC,*** p<0.0001 and UM vs HC, **p=0.0088, Dunn’s multiple comparison). G ro u p s H R P 2 c o n c . ( n g / m l ) C M U M H C 0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 1 2 0 0 0 *** *** ** Figure 4. 14 HRP2 levels in the study groups at baseline *** p<0.0001 ** p<0.001 University of Ghana http://ugspace.ug.edu.gh 96 4.6.2 Time course levels of HRP2 in the study groups Time course levels of HRP2 were determined in CM and UM patients (Figure 4.15). HRP2 levels dropped significantly (p<0.0001, Kruskal Wallis) in both CM and UM. Levels dropped below detection levels fourteen days after recovery from coma and seven days after initial presentation in CM and UM respectively. G ro u p s H R P 2 c o n c . ( n g / m l ) D a y 0 R e c D a y 7 D a y 1 4 D a y 0 D a y 7 D a y 1 4 0 2 5 0 0 5 0 0 0 7 5 0 0 1 0 0 0 0 C M U M* * * *** Figure 4. 15 Time course HRP2 levels in the study groups *** p<0.0001 University of Ghana http://ugspace.ug.edu.gh 97 4.7 Protease/Chemokine Levels 4.7.1 Baseline levels of MMP9 in the study groups MMP9 levels were determined in the various study group at baseline (Figure 4.16). UM showed the highest MMP9 levels (p<0.0001, Kruskal Wallis) compared with both CM and HC (CM vs UM, **p=0.025, CM vs HC, p>0.999 and UM vs HC, **p=0.031, Dunn’s multiple comparism). C M U M h c 0 2 0 0 4 0 0 6 0 0 G ro u p s M M P 9 c o n c . ( n g / m l Figure 4. 16 MMP9 levels in the study groups at baseline University of Ghana http://ugspace.ug.edu.gh 98 4.7.2 Time course levels of MMP9 in the study groups Time course measurement of MMP9 levels in the various study groups were done (Figure 4.17). Plasma levels of MMP9 in CM patients were not significantly different at initial clinical presentation compared to subsequent time points (p>0.05, Kruskal Wallis). UM showed higher MMP9 levels (***p<0.05) at initial presentation compared to seven and fourteen days after initial presentation. (Day 0 vs Day 7: p=0.0013. Day 0 vs Day 14; p=0.03, Dunn's multiple comparisons). G ro u p s M M P 9 c o n c . ( n g / m l D a y 0 R E C D a y 7 D a y 1 4 D a y 0 D a y 7 D a y 1 4 H C 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 *** C M U M H C Figure 4. 17 Time course MMP9 levels in the study groups *** p<0.0001 University of Ghana http://ugspace.ug.edu.gh 99 4.7.3 Baseline levels of SDF-1 in the study groups SDF-1 levels were determined in the various study group at baseline (Figure 4.18). SDF-1 levels were not significantly different in the initial evaluation between study groups (p>0.05, Kruskal Wallis). C M U M H C 0 1 0 0 0 2 0 0 0 3 0 0 0 G ro u p s S D F - 1 c o n c ( n g / m l ) Figure 4. 18 SDF-1 levels in the study groups at baseline University of Ghana http://ugspace.ug.edu.gh 100 4.7.4 Time course levels of SDF-1 in the study groups Time course plasma levels of SDF-1 (Figure 4.19) did not show any significantly difference (p>0.05, Kruskal Wallis) within and between study groups. Levels in CM and UM were not significantly different from healthy individuals (p>0.999, Kruskal Wallis). G ro u p s S D F - 1 c o n c ( n g / m l ) D a y 0 R E C D a y 7 D a y 1 4 D a y 0 D a y 7 D a y 1 4 H C 0 1 0 0 0 2 0 0 0 3 0 0 0 C M U M H C Figure 4. 19 Time course SDF-1 levels in the study groups University of Ghana http://ugspace.ug.edu.gh 101 CHAPTER FIVE 5 DISCUSSION, CONCLUSION AND RECOMMENDATIONS 5.1 Discussion Sequestration of infected red blood cells (iRBCs) in the brain microvasculature has been shown to be the hallmark of CM and the resultant damage to the endothelium has been postulated as a major initiator of CM (Cooke et al., 2000; Silamut et al., 1999a; Taylor et al., 2004b; Weatherall et al., 2002). The role of cEPCs in the repair of damaged microvasculature has been studied extensively in diseases associated with microvascular damage. The extension of the role of cEPC and factors that affect their levels and function were assessed in this study. The study was carried out in collaboration from five major referral hospitals in the Greater Accra Region of Ghana. A total of 50 children presenting with CM to these hospitals were recruited and most of these children were aged between a year and twelve years. The number of cases could be indicative of the incidence of CM within this region of Ghana as most cases of CM are referred to these five collaborating hospitals. These hospitals are within a radius of 4 miles from the research centre where analysis of blood samples from the patients was done. The study could not include hospitals from farther distances as processing of samples for rare cells considered in the study need to be done within 2-3 hours after collection. To assess the possibility of extending the recruitment of study participants to remote centers in the future, the study initially evaluated methods that would allow preservation of samples for longer periods for delayed processing. University of Ghana http://ugspace.ug.edu.gh 102 CECs and EPCs have been shown as surrogate markers of vascular injury and repair respectively and the successful preservation of these rare cells in biological samples for delayed flow cytometry is very critical for evaluation, particularly in diseases characterized by endothelial damage and interventional clinical trials (Bogoslovsky et al., 2013). It has been recommended that flow cytometric analysis of CECs and EPCs be done within 2-3 hours of collection of samples (Duda et al., 2007). However, not much cryopreservation protocols for EPCs and CECs are available which would fulfill the requirements for multicenter trials. This study therefore evaluated the ability of Cyto-chex BCT (C-C BCT), a commercially available blood cell preservative, to either preserve or stabilize these cells and other immune markers for delayed flow cytometry. Generally, whole blood preserved in Cyto-chex BCT at 40C maintained the overall discrimination of the major leukocyte populations (lymphocytes, monocytes, granulocytes) making gating on the groups possible. This was evident from whole blood stored in C-C BCT at 40C for seven days, where lymphocytes, monocytes and granulocytes could be clearly differentiated from each other as was observed in the case of whole blood stored in EDTA on day 0 (Figure 4.3). EDTA is the recommended anticoagulant for the enumeration of CECs and EPC (Duda et al., 2007). EDTA on day seven showed clumped cells, making further evaluation of subpopulation of leukocyte impossible. A study by Warrino et al. (2005) also reported the ability to differentiate the various leukocyte populations after seven days of storage of whole blood in C-C BCT at room temperature. Other preservatives such as Transfix (Cytomark, Buckingham, UK) have shown similar leukocyte discriminatory characteristics after seven days of storage (Hoymans et al., 2012). University of Ghana http://ugspace.ug.edu.gh 103 Flow cytometric analysis of EPCs showed that, these rare cells were stable in whole blood stored in C-C BCT for at least 48 hours (2 days) at 4oC. This is evident in the fact that day 2 levels of EPCs in C-C BCT were not significantly different from that in EDTA at baseline (p<0.05) and earlier time points in C-C BCT (Days 0 and 1) [Figure 4.4]. Significant decrease of viable EPC numbers have been shown in blood samples stored in EDTA for more than 24 hours (Masouleh et al., 2010). Cryopreservation of PBMC at -160°C , -80°C and short-term preservation at room temperature have shown decreased EPC numbers (Bogoslovsky et al., 2015). However, the stability of EPCs have also been demonstrated in other stabilizing agents such as the Transfix cellular stabilizing reagents (Hoymans et al., 2012). C-C BCT has therefore shown promise in preserving EPCs. CECs on the other hand have been used as a marker for endothelial damage and their presence in peripheral blood is indicative of endothelial damage (Dignat‐George and Sampol, 2000). Even though their half-life is not known in normal individuals and patients (Erdbruegger et al., 2006), data from this study shows the stability of CECs in C-C BCT for at least 48 hours (Figure 4.5). There was a significant drop (*p<0.023) in CEC levels on day 7 in C-C BCT (0.0004%) compared with that in EDTA at baseline (0.0026%). Data from the study therefore suggest the ability of C- C BCT to extend the processing time beyond the 3 hours recommended by Duda et al., (2007). The stability of some individual receptors expressed on circulating endothelial cells and their progenitors were also evaluated for seven days in C-C BCT. CD309 (VEGFR2) and CD133 which are markers for mature and immature endothelial progenitor cells respectively were stable for at least 2 days in C-C BCT (Figure 4. 5). Mean levels of these cells in the first 48 hours in C-C BCT University of Ghana http://ugspace.ug.edu.gh 104 were not significantly different from that in EDTA at baseline (p<0.05). Other markers for endothelial cells such as CD34 and CD31 were stable for at least seven days in C-C BCT. In other studies, overnight storage at 4°C did not have any effect on CD34+ cell counts and storage in liquid nitrogen for 7 weeks did not affect the percentage of CD34+ cells but was associated with a 26% drop in cell viability (Zubair et al., 2010). Pan leukocyte markers such as CD45 and CD11b were stable for at least 24 and 48 hours respectively (Figure 4.7). This shows the ability of C-C BCT to stabilize these immune epitopes for delayed analysis. CD45 has been shown to be stable in C-C BCT for seven days at room temperature (Warrino et al., 2005). Stability of some common immune markers in C-C BCT were evaluated. Immune markers such as CD4, CD8, CD19 and CD16 have all been shown to be stable in C-C BCT for at least seven days after phlebotomy (Warrino et al., 2005). CD4 and CD8 which are also part of the HIV panel of markers were shown to be stable at least seven days in C-C BCT in this study (Warrino et al., 2005). These cells are usually suitable for analysis within 72 hours of collection in either EDTA or Heparin anticoagulant (Mandy et al., 2003). However, in general, cells are considered suspect if they are analyzed after 48 hours of collection (Bergeron et al., 2002). Other receptors such as CD15 mostly found on granulocytes and CD14, also common on monocytes and macrophages were shown to be stable in C-C BCT at least 7 and 2 days respectively. The stability of these common individual receptors in C-C BCT therefore shows promise in delayed analysis. C-C BCT was however not utilized in current study as all affiliated hospitals were within an hour reach by ground transportation. University of Ghana http://ugspace.ug.edu.gh 105 Based on studies by Gyan et al. (2009), which associated decreased cEPC levels to CM in Ghanaian children, this current study hypothesized that recovery from CM is associated with increased cEPC levels and UM patients who progress on the continuum to develop CM will have decreasing levels of cEPCs. Also CM patients who die may have decreasing levels of cEPC. The study therefore evaluated the time course of the host response to P. falciparum–induced microvascular damage. Levels of cEPC were determined at baseline and different times during recovery in CM and UM patients who reported to the five collaborating hospitals and HC from community schools around these hospitals. Pairwise comparison of baseline levels of cEPC in the various study groups indicated lower levels (0.042%) in CM compared to UM (0.140%) and HC (0.083%) [Figure 4.9]. This is consistent with earlier studies by Gyan et al. (2009) where cEPCs defined as the dual expression of CD34+/VEGFR2+ and CD34+/CD133+ for immature EPC, were higher in both asymptomatic and uncomplicated malaria cases. Several studies have shown that recruitment of EPC represents the body’s ability to balance vascular damage with repair (Asahara et al., 1999; Hill et al., 2003; Yoder et al., 2007). A failed attempt to produce and recruit enough EPCs to sites of endothelial injury caused by the sequestration of iRBCs could account for the disease state seen in CM. Levels of cEPCs have been shown to decrease in disease conditions characterized by endothelial damage such as cardiovascular disease, diabetes and hypertension (Vasa et al., 2001; Verma and Anderson, 2002). Time course evaluation of levels of cEPCs in CM patients showed a sharp increase at recovery (i.e. BCS>3) [0.117%] and at day 7 post recovery (0.157%) compared with levels at baseline University of Ghana http://ugspace.ug.edu.gh 106 (0.042%) [Figure 4.10]. This trend supports the study hypothesis that CM patients who recover may have increased cEPC levels. The increased levels at recovery and post recovery suggest that the host attempts to repair microvascular damage caused by the activation of the endothelium following sequestration of the malaria parasites. It has been shown that the balance between endothelial injury and repair is critical for the maintenance of endothelial integrity (Sabatier et al., 2009). Endothelial activation has been demonstrated in both mild and severe malaria even though it increases with disease severity (Park et al., 2012). Higher cEPC levels at onset of uncomplicated malaria and the maintenance of levels at sampling time points seem to promote a balance between endothelial damage and repair. UM that converts to CM could be associated with decreasing cEPC levels. However, data on patients prior to conversion to CM could not be obtained in this study. In some in vitro studies, CECs have been reported to inhibit the proliferation and migration of EPCs thus affecting the functional capacity of endothelial repair (Holmén et al., 2005). CECs are a reliable marker of microvascular injury and in most cases high levels of these cells are associated with endothelial activation and damage (Dignat‐George and Sampol, 2000). These cells are usually rare in normal individual and higher in individuals who are acutely ill. CM which is characterized by sequestration and subsequent damage to the endothelium saw high levels of CECs at initial presentation compared to UM and HC. This supports the study hypothesis that CM would have higher CECs due the detachment of endothelial cells resulting from endothelial damage. Studies have found a correlation between CECs and other markers of endothelial activation such as von Willebrand factor and plasma tissue factor (Makin et al., 2004a). Levels of these endothelial activation markers have been found to be elevated in CM (de Mast et al., 2007; Mohanty et al., 1997). University of Ghana http://ugspace.ug.edu.gh 107 Longitudinal quantitation of CECs in other conditions showed that their levels vary according to the clinical evolution with patients in clinical remission or in recovery phases having reduced levels. It is therefore expected that levels of CEC would decrease as patients recover. However, data from this study showed CM patients having increasing levels of CEC even at fourteen days post recovery form coma, contrary to what was expected. In CM, the endothelium is known to be activated as a result of increased levels of activation markers such as vWF and subsequent endothelial injury or damage. These injured cells are detached from the endothelium, thereby increasing the levels of CECs in peripheral blood as was observed at initial presentation of CM in this study. Not all injured cells are detached immediately and may need to be replaced gradually during clinical remission. This may increase CEC levels in peripheral blood during the time course of recovery from CM as seen in Figure 4.9. The gradual replacement of damaged endothelial cells is further explained by the increased levels of bone marrow derived cEPC in the time course of CM (known to repair and replace damaged ECs in CM time course) [Figure 4.7]. CEC levels in UM patients saw a sharp increase seven days after initial malaria diagnosis (Figure 4.9) suggesting a possible levels of endothelial injury at the onset of Plasmodium infection. However, due to high levels of cEPC in UM at baseline (Figure 4.6) there was the ability to balance damage and repair effectively. The presence of the parasite could possibly cause endothelial activation and the endothelial cells affected may have to be replaced with time. The high cEPC in UM permits effective displacement of possible injured cells into circulation resulting in increased CECs levels in UM on day seven after initial presentation. Observation of CEC levels in UM in University of Ghana http://ugspace.ug.edu.gh 108 this study is consistent with other studies that have shown endothelial disturbance in malaria infection (Boubou, 2000; de Mast et al., 2007; Ohnishi, 1999; Park et al., 2012). However, microvascular damage has been shown to induce the expression of chemokine/proteases such as SDF-1 and MMP9 (Huang et al., 2009; Tilling et al., 2009). These molecules are involved in the mobilization, proliferation and migration of EPCs to the sites of endothelial damage. Their levels are expected to be higher in conditions associated with endothelial damage such as CM. To mobilize cEPCs for endothelial repair, CM patients were expected to have high levels of SDF-1 and MMP9. However, data from the study could not significantly show any differences in the serum SDF-1 levels at baseline and time course in the three study groups. UM and HC had higher levels (though not significant) than CM contrary to what was observed in earlier studies by Gyan et al. (2009). MMP9 on the other hand was significantly higher in UM compared to CM and HC. Time course data showed a reduction in the MMP9 levels in UM at day seven post initial presentation whiles CM showed no significant difference. Data suggest the attempt by UM patients to mobilize and migrate EPCs at initial presentation. This attempt to mobilize and migrate EPC seems to be lacking or delayed in CM. Levels of these markers represents that of peripheral or venous blood and may be different from levels close to site of damage. CM could have higher levels of MMP 9 and SDF- 1 at or close to the site of endothelial damage as EPCs are needed at these sites. However, more studies need to be done to assess the dynamics of these markers and their function in microvascular damage and repair. University of Ghana http://ugspace.ug.edu.gh 109 Dysregulation of Angiopoietins has been shown in CM (Conroy et al., 2009). In a stable state, the survival and activation of ECs is regulated by Angiopoietin 1 through the Tie-2 receptor whereas Ang-2 opposes this process (Conroy et al., 2012). Ang-2 antagonizes Ang-1 by sensitizing the endothelium to inflammation and increasing the expression of receptors that bind to iRBCs (Shikani et al., 2012). Higher levels of Ang-2 and lower levels of Ang-1 have been observed to correlate with severe malarial infections (Conroy et al., 2009; Lovegrove et al., 2009; Yeo et al., 2008). This study showed higher plasma levels of Ang-2 at initial presentation of cerebral (5260pg/ml) compared to uncomplicated malaria (4041pg/ml) and healthy children (4909pg/ml) even though these were not statistically significantly different (p=0.12, Kruskal Wallis test). Time course level of Ang-2 in CM showed an increase during the coma state and was unstable at subsequent time points. UM on the other hand showed a gradual increase in the time course. Ang-1 levels were observed to be higher in CM (17021pg/ml) than in UM (9248pg/ml) contrary to earlier studies by Conroy et al. (2009) and Lovegrove et al. (2009). Levels of Ang-1 seem to be time dependent as there was a sharp drop (8967pg/ml) in the levels at recovery. Therefore analyzing Ang-1 levels at early stage of coma and at late stage of coma in cerebral malaria could show significant differences. This could possibly explain the differences between the Ang1 levels in this study and that in studies by Conroy et al. (2009) and Lovegrove et al. (2009). Studies relating to brain injury have also reported decreased abundance of Ang-1 and an up-regulation of Ang-2 in the early phase post brain injury (Chittiboina et al., 2013). An increase in Ang-1 levels and a corresponding decrease in Ang-2 seven days post recovery correlated with complete recovery form coma (BCS =5) in the CM patients. The time course levels in Ang-1 in CM also University of Ghana http://ugspace.ug.edu.gh 110 confirm the antagonistic effects of Ang-1 and Ang-2 as indicated by earlier studies (Conroy et al., 2009; Lukasz et al., 2008; Parikh, 2013). The ratio of Ang-2 and Ang-1 in this study, did not show any statistical significance (p>0.05) at initial clinical evaluation of CM and UM as well as HC. However, a sharp increase (day 0 vs recovery, p<0.005, Kruskal Wallis and Dunn’s multiple comparism) in the ratio of these two biomarkers in the late stage of coma (i.e. early stage of recovery from coma) could predict some level of endothelial injury in the CM patients. The decrease in the ratio seven days post recovery indicated resolution of the injury. Ratios in UM patients were not significantly different (p>0.05) in the time course of disease resolution. Some studies have reported the ability of Ang1:Ang2 ratio to discriminate between severe and uncomplicated malaria (Conroy et al., 2009; Lovegrove et al., 2009). The time course evaluation of these endothelial mediators in CM has demonstrated their importance in the resolution of severe malaria in African children. Lovegrove et al. (2009) reported the ability of Ang-1 and Ang-2 to discriminate CM and UM in Thai adults. Data from this study and others have therefore, demonstrated the importance of levels of these two biomarkers in the resolution of severe malaria. Due to the upregulation of some markers of endothelial damage/dysfunction and brain micro haemorrhages in CM, some studies have suggested a possible clotting disorder (Moxon et al., 2013) which is still being debated. Moxon et al. (2013) showed that decreased expression of the anticoagulant and protective receptors TM and EPCR in the brain endothelium make it particularly vulnerable to injury. Cell-surface expression of TM is also known to be reduced when the endothelium is activated, leading to shedding of the molecule and subsequent increase in soluble University of Ghana http://ugspace.ug.edu.gh 111 forms (Faust et al., 2001). Soluble TM has therefore been proposed as both a diagnostic and prognostic marker of endothelial activation/dysfunction (Page and Liles, 2013). In the current study, soluble TM levels were significantly higher (p<0.05) in CM patients compared to UM and HC. Activation of the endothelium has already been established in CM and higher levels of sTM in CM observed may be due to shedding of this anticoagulant which is present in large quantities on the surface of the endothelium in microcirculation. This is consistent with other markers of activation such as CECs and Angiopoietin 2 as discussed earlier and other pro-inflammatory cytokines and anaemia (Maya et al., 2008). Other studies have shown that higher sTM correlated with disease severity (Butthep et al., 2006; Ikegami et al., 1998; Kinasewitz et al., 2004). The current study is consistent with observation in other studies which have reported higher levels of sTM in children with severe than uncomplicated malaria and uninfected healthy controls (Mita- Mendoza et al., 2013; Ohnishi, 1999; Page and Liles, 2013). Like thrombomodulin, EPCR is expressed on all vascular endothelial cells and is involved in activating protein C by stabilizing the interaction of protein C with the thrombin–thrombomodulin complex (Moxon et al., 2009). It is expressed in lower level in micro-vessels of the brain (Laszik et al., 1997) but upon activation, they are shed from the endothelium, thereby increasing its plasma levels. It has been reported that shedding of this molecule leads to low expression on the endothelium and increase plasma levels makes the brain vulnerable (Moxon et al., 2013), thus implicating EPCR in the pathology associated with CM. The current study show that the means levels of sEPCR in CM (20.14pg/ml) was higher (even though not statically significant, p>0.05, Kruskal-Wallis test) compared with UM (16.82pg/ml) and HC (15.39pg/ml). Other studies have reported reduced expression and increased plasma levels of these EPCR in bacterial sepsis (Faust University of Ghana http://ugspace.ug.edu.gh 112 et al., 2001), dengue haemorrhagic fever (Cabello-Gutiérrez et al., 2009) and Crohn’s disease (Scaldaferri et al., 2007). The definition of CM includes the inability of a child or patient to localize a painful stimulus, presence of peripheral asexual P falciparum parasitaemia with no other identified causes of encephalopathy. However, in malaria-endemic regions such as Ghana, asymptomatic parasitaemia is common and children in coma who show positive peripheral parasitaemia are often initially considered as having CM. On the other hand, some children or patients in coma without peripheral parasitaemia (as a result of parasite sequestration) may not be considered as having CM. These false positives and false negatives often results in misdiagnosis and subsequent implications for patient care. Retinopathy which consist of two unique features: patchy retinal whitening and focal changes of vessel colour has been shown to be highly specific for encephalopathy of malarial etiology (Beare et al., 2011). However, detection of retinopathy requires highly trained personnel and expensive equipment (Seydel et al., 2012). A more user-friendly biomarker, HRP2, has been shown to predict iRBC sequestration and a quantitative measure of plasma levels of this P. falciparum specific protein have been evaluated and used to discriminate CM from other forms of malaria and also CM and non-malaria comatose conditions (Kariuki et al., 2014; Seydel et al., 2012). The current study also evaluated the plasma levels of HRP2 in CM, UM and HC and showed that CM patients had the highest levels of HRP2 (2281ng/ml [95% CI 1453-3109]) compared with UM (353ng/ml [95% CI 162-543]). Healthy controls did not have any detectable HRP2. This result is consistent with other studies that detected higher HRP2 levels in CM compared with UM (Kariuki University of Ghana http://ugspace.ug.edu.gh 113 et al., 2014; Seydel et al., 2012). Seydel et al., 2012, upon determining the plasma levels of HRP2 in Malawian children who had histological evidence of sequestration (through autopsy), proposed a cutoff of >1700ng/ml as sensitive and specific for CM. Even though 42% of CM cases recruited for this current study had HRP2 levels above the cutoff proposed by Seydel et al., 2012, the study could not confirm this cutoff in Ghanaian children as autopsies were not conducted on cases that died. The level of endemicity may affect the cutoff levels of HRP2 and therefore estimation of cutoff values in different endemic areas may be very critical. 5.2 Conclusion Combined measurements of the endothelial indicators evaluated in this study offers a non-invasive and user friendly approach to assess endothelial integrity in CM patients and also offers biomarker strategies that could predict who is at risk of developing CM. These indicators also show the potential to monitor response to treatment by assessing the balance between damage and repair as an index of the endothelial integrity. This study has shown that low cEPC, high CEC, high sTM, and high HRP2 levels were associated with cerebral malaria. An increase in cEPC levels has been shown to be very critical in the resolution of coma in CM patients. CECs and cEPCs levels could predict degree of endothelial activation and/or damage in CM. Lower levels of MMP9 in CM indicate reduced mobilization and proliferation of EPC in CM. This study also showed Angiopoietins as promising endothelial mediators and good biomarkers predicting endothelial damage and repair. Soluble TM levels were elevated in CM as a result of University of Ghana http://ugspace.ug.edu.gh 114 shedding from the endothelium and therefore have been shown as good prognostic and diagnostic markers in predicting endothelial damage in CM. Soluble EPCR has also shown promise as biomarker in predicting CM and therefore its role in CM pathogenesis cannot be overlooked. This study confirms HRP2 as a biomarker capable of differentiating CM from UM and other non- malaria comatose conditions. The utility of HRP2 as an alternative to retinopathy and a more user friendly marker in confirming CM is highly supported by this study. C-C BCT has shown the ability to preserve rare cells such as EPC, CECs and other immune markers. Extension of this technique is therefore possible for the preservation of biological samples from remote settings where flow cytometers may not be readily available. 5.3 Recommendations Further studies that would assess the migratory capacity and the functionality of EPCs and other markers involved in the repair of damaged endothelium and techniques that could assess levels of markers at the site damage (especially, the brain vasculature in CM) are highly recommended. Data from this studies show peripheral or systemic levels of this markers. Further studies targeting levels of these markers and several others at sites of endothelial damage would be of great benefit to the understanding of the pathogenesis of cerebral malaria. University of Ghana http://ugspace.ug.edu.gh 115 Therapies that would increase levels of EPCs and other markers which have shown promise in the resolution of CM could be considered. Consideration of EPCs in malaria vaccines cannot also be overemphasized. 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Thrombospondin binds falciparum malaria parasitized erythrocytes and may mediate cytoadherence. Nature 318, 64-66. Rock, E. P., Roth, E. J., Rojas-Corona, R. R., Sherwood, J. A., Nagel, R. L., Howard, R. J., and Kaul, D. K. 1988. Thrombospondin mediates the cytoadherence of Plasmodium falciparum- infected red cells to vascular endothelium in shear flow conditions. Blood 71, 71-75. Roll Back Malaria 2003. Economic costs of malaria. In World Health Organization. Available from: URL: http://www. rbm. who. int/cmc_upload/0/000/015/363/RBMInfosheet_10. htm. Roll Back Malaria 2013. Key Malaria Facts, 2012. In. Rowe, A., Obeiro, J., Newbold, C. I., and Marsh, K. 1995. Plasmodium falciparum rosetting is associated with malaria severity in Kenya. Infection and Immunity 63, 2323-2326. University of Ghana http://ugspace.ug.edu.gh 136 Rowe, J. A., Claessens, A., Corrigan, R. A., and Arman, M. 2009. 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IH, Basic Malaria Microscopy, Part I. Learner's Guide. In, (Basic Malaria Microscopy). World Health Organization 2012. World Malaria Report 2010. In World Health Organization Geneva. Availabe http://www. who. int/malaria/world_malaria_report_2010/en/index. html. World Health Organization 2014a. Malaria. WHO Fact sheet N 94, Updated March 2014. In. World Health Organization 2014b. WHO Global Malaria Programme. World Malaria Report 2014. In, (WHO Press, Geneva, Switzerland). World Health Organization 2014c. World Malaria Report 2014 (2014). In WHO: Geneva. Woywodt, A., Blann, A. D., Kirsch, T., Erdbruegger, U., Banzet, N., Haubitz, M., and Dignat- George, F. 2006. Isolation and enumeration of circulating endothelial cells by immunomagnetic isolation: proposal of a definition and a consensus protocol. Journal of Thrombosis Haemostasis 4, 671-677. Woywodt, A., Streiber, F., de Groot, K., Regelsberger, H., Haller, H., and Haubitz, M. 2003. Circulating endothelial cells as markers for ANCA-associated small-vessel vasculitis. The Lancet 361, 206-210. Wu, H., Chen, H., and Hu, P. C. 2007. Circulating endothelial cells and endothelial progenitors as surrogate biomarkers in vascular dysfunction. Clinical Laboratory 53, 285. Yancopoulos, G. D., Davis, S., Gale, N. W., Rudge, J. S., Wiegand, S. J., and Holash, J. 2000. Vascular-specific growth factors and blood vessel formation. Nature 407, 242-248. Yeo, T. W., Lampah, D. A., Gitawati, R., Tjitra, E., Kenangalem, E., Piera, K., Price, R. N., Duffull, S. B., Celermajer, D. S., and Anstey, N. M. 2008. Angiopoietin-2 is associated with University of Ghana http://ugspace.ug.edu.gh 142 decreased endothelial nitric oxide and poor clinical outcome in severe falciparum malaria. Proceedings of the National Academy of Sciences 105, 17097-17102. Yoder, M. C. 2009. Defining human endothelial progenitor cells. Journal of Thrombosis and Haemostasis 7, 49-52. Yoder, M. C., Mead, L. E., Prater, D., Krier, T. R., Mroueh, K. N., Li, F., Krasich, R., Temm, C. J., Prchal, J. T., and Ingram, D. A. 2007. Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood 109, 1801-1809. Zubair, A. C., Malik, S., Paulsen, A., Ishikawa, M., Mccoy, C., Adams, P. X., Amrani, D., and Costa, M. 2010. Evaluation of mobilized peripheral blood CD34+ cells from patients with severe coronary artery disease as a source of endothelial progenitor cells. Cytotherapy 12, 178-189. University of Ghana http://ugspace.ug.edu.gh 143 APPENDICES Appendix 1: Buffers and Reagents A. Buffers for ELISA (Buffer preparation was the same for all the biomarkers) I. Coating buffer 90g of NaCl plus 10.9g of Na2HPO4 (dibasic) plus 3.2g of NaH2PO4 (monobasic) in 1000ml of double distilled water. II. Washing buffer 0.05% Tween 20 in PBS. III. Blocking buffer 1% BSA plus 0.05% Tween 20 in PBS. IV. Reagent diluent 1% BSA in PBS. B. Giemsa Buffer for parasite staining Na2HPO4 1.0g KH2PO4 0.7g Distilled water 1 litre (Adjusted pH, 7.25; Temp. 30.50C) C. Sickling test Buffer Na2S2O5 2% of Na2S2O5 in distilled water. D. Antibody and Fluorochromes Item Fluorochrome Supplier: Mouse Anti-Human CD15 FITC BD Pharmingen Mouse Anti-Human CD 14 PE BD Pharmingen Human CD 45 Antibodies FITC Becton Dickinson Human CD133/2 (AC141) Antibodies PE Miltenyi Biotec (MACS) Human CD 34 Antibodies PerCP Becton Dickinson Human CD 31 Antibodies APC Miltenyi Biotec (MACS) Human CD 11b Antibodies FITC Miltenyi Biotec (MACS) Anti-Human CD 309 (VEGF R2/KDR) APC Miltenyi Biotec (MACS) ChromPure mouse IgG1 Whole molecule Size 5.0mg Jackson ImmnoResearch Labs University of Ghana http://ugspace.ug.edu.gh 144 Appendix 2A: Consent for Children with Malaria Title: Circulating endothelial cells and the pathogenesis of malaria Principal Investigator: Ben Gyan, PhD Address: Department of Immunology, NMIMR, Box LG 581, Legon Information: (To be read or translated to parents/guardians in their own mother tongue) Dear Volunteer, This consent form contains information about the research entitled Circulating endothelial cells and the pathogenesis of malaria. In order to be sure that you are informed about being in this research, we are asking you to read (or have read to you) this Consent Form. You will also be asked to sign it (or make your mark in front of a witness). We will give you a copy of this form. This consent form might contain some words that are unfamiliar to you. Please ask us to explain anything you may not understand. Why this study is planned Your child is being asked to participate in the above study in order to find out factors in the blood that may be of risk to severe malaria. Malaria is caused by a germ that is passed from one person to the other by the bite of a mosquito that carries the malaria germ. Malaria is a very serious health problem in Ghana, as it is in many African countries. We do not know why some children become severely ill from malaria or why some of those children die from malaria. To understand this problem we need to study children who come to the hospital with severe malaria and compare them to children who have less severe malaria, and to other children who are feeling well. The purpose of the study is to find out what factors they already have in their blood that may make University of Ghana http://ugspace.ug.edu.gh 145 them severely sick when they have malaria. If we can find the answer to this question, we hope to be able to suggest new ways of controlling such severe sicknesses in malaria. General Information and your part in the study For a child to qualify to be part of this study that child should be between the ages of 1 and 12 years. If your child/ward agrees to be in the study, we will collect venous blood sample for laboratory diagnosis and 2 ml (teaspoonful) for our research at the time of admission, 7 days and 14 days after recovery. If you miss a scheduled follow-up visits (7 days and 14 days after recovery), we may contact you at home by phone, or in person to schedule another visit and to see if you still want to take part in the research. When this contact is made you will not be identified as being in this research. Possible Benefits There are no direct benefits to your child from this study. However, his/her participation may help us develop better malaria treatment. He/she will not be paid for participation in this study but you will be reimbursed with an amount of fifteen Ghana cedis for your time and travel during the follow up visits. Possible Risks The amount of blood collected is harmless, although there may be a slight pain and bruising at the bleeding site. All subjects will receive appropriate treatment as necessary. Sterile techniques and disposable, single-use equipment will be used at all times. University of Ghana http://ugspace.ug.edu.gh 146 Withdrawal from study We would like to stress that this study is strictly voluntary. Should the child decide not to participate; it will have no consequences for him/her. Should the volunteer, at any point during the study, decide that he/she do not wish to participate any further, you are free to terminate the participation, effective immediately. Any such decision will be respected without any further discussion. Your decision will not affect the health care you would normally receive. Visits If the child misses a scheduled visit, we may contact you at home by phone, or in person to schedule another visit and to see if you still want to take part in the research. When this contact is made you will not be identified as being in this research. Confidentiality All information gathered would be treated in strict confidentiality. We will protect information about your child taking part in this research to the best of our ability. The child will not be named in any reports. However, the staff of [list all groups that may access the research records] may sometimes look at his/her research records. If you have any questions, please feel free to ask the physician in charge. Someone from the IRB or Ethical Committee might want to ask you questions about being in the research, but you do not have to answer them. A court of law could order medical records shown to other people, but that is unlikely. Contacts: If you ever have any questions about the research study or study-related problems, you may contact Prof. Ben Gyan of the Noguchi Memorial Institute for Medical Research (0244 University of Ghana http://ugspace.ug.edu.gh 147 726016) at any time. For questions about the ethical aspects of this study or your rights as a volunteer, you may contact Dr. Chris Dadzie, Chairman, Institutional Review Board, NMIMR, University of Ghana (0302 501178/9) or Chairman of the Ghana Health Service Ethical Committee (Tel. 0302 681109) Your rights as a participant This research has been reviewed and approved by the NMIMR IRB and Ghana Health Service Ethical Committee. An IRB or Ethical Committee is a committee that reviews research studies in order to help protect participants. If you have any questions about your rights as a research participant you may contact [Dr. Chris Dadzie, Tel 0302-501-178/179 or Chairman of the Ghana Health Service Ethical Committee (Tel. 0302 681109) VOLUNTEER AGREEMENT The above document describing the benefits, risks and procedures for the research title Circulating endothelial cells and the pathogenesis of malaFigure 1.1 Model of the development and resolution of cerebral malaria ........................................................................................................................................... 27 ria has been read and explained to me. I have been given an opportunity to have any questions about the research answered to my satisfaction. I agree my child/ward to participate as a volunteer. ------------------------------------------------ ------------------------------------------------ Date Signature or thumbprint of volunteer If volunteer’s Parent/Guardian cannot read the form themselves, a witness must sign here: University of Ghana http://ugspace.ug.edu.gh 148 I was present while the benefits, risks and procedures were read to the volunteer. All questions were answered and the volunteer’s Guardian/Parent has agreed to take part in the research. ------------------------------------------------ ----------------------------------------------- Date Signature or thumbprint of witness I certify that the nature and purpose, the potential benefits, and possible risks associated with participating in this research have been explained to the above individual. ------------------------------------------------ ----------------------------------------------------- Date Signature of person who obtained consent University of Ghana http://ugspace.ug.edu.gh 149 Appendix 2B: Consent for Healthy Controls Title: Circulating endothelial cells and the pathogenesis of malaria Principal Investigator: Ben Gyan, PhD Address: Department of Immunology, NMIMR, Box LG 581, Legon Information: (To be read or translated to parents/guardians in their own mother tongue) Dear Volunteer, This consent form contains information about the research entitled Circulating endothelial cells and the pathogenesis of malaria. In order to be sure that you are informed about being in this research, we are asking you to read (or have read to you) this Consent Form. You will also be asked to sign it (or make your mark in front of a witness). We will give you a copy of this form. This consent form might contain some words that are unfamiliar to you. Please ask us to explain anything you may not understand. Why this study is planned Your child is being asked to participate in the above study in order to find out factors in the blood that may be of risk to severe malaria. Malaria is caused by a germ that is passed from one person to the other by the bite of a mosquito that carries the malaria germ. Malaria is a very serious health problem in Ghana, as it is in many African countries. We do not know why some children become severely ill from malaria or why some of those children die from malaria. To understand this problem we need to study children who come to the hospital with severe malaria and compare them to children who have less severe malaria, and to other children who are feeling well. The purpose of the study is to find out what factors they already have in their blood that may make University of Ghana http://ugspace.ug.edu.gh 150 them severely sick when they have malaria. If we can find the answer to this question, we hope to be able to suggest new ways of controlling such severe sicknesses in malaria. General Information and your part in the study For a child to qualify to be part of this study that child should be between the ages of 1 and 12 years. If your child/ward agrees to be in the study, we will collect venous blood sample for laboratory diagnosis and 2 ml (teaspoonful) for our research initially and 7 days and 14 days later. If you miss a scheduled follow-up visits (7 days and 14 days) in your school, we may contact you at home by phone, or in person to schedule another visit and to see if you still want to take part in the research. When this contact is made you will not be identified as being in this research. Possible Benefits There are no direct benefits to your child from this study. However, his/her participation may help us develop better malaria treatment. He/she will not be paid for participation in this study but you will be reimbursed with an amount of fifteen Ghana cedis for your time and travel during the follow up visits. Possible Risks The amount of blood collected is harmless, although there may be a slight pain and bruising at the bleeding site. All subjects will receive appropriate treatment as necessary. Sterile techniques and disposable, single-use equipment will be used at all times. University of Ghana http://ugspace.ug.edu.gh 151 Withdrawal from study We would like to stress that this study is strictly voluntary. Should the child decide not to participate; it will have no consequences for him/her. Should the volunteer, at any point during the study, decide that he/she do not wish to participate any further, you are free to terminate the participation, effective immediately. Any such decision will be respected without any further discussion. Your decision will not affect the health care you would normally receive. Visits If the child misses a scheduled visit, we may contact you at home by phone, or in person to schedule another visit and to see if you still want to take part in the research. When this contact is made you will not be identified as being in this research. Confidentiality All information gathered would be treated in strict confidentiality. We will protect information about your child taking part in this research to the best of our ability. The child will not be named in any reports. However, the staff of [list all groups that may access the research records] may sometimes look at his/her research records. If you have any questions, please feel free to ask the physician in charge. Someone from the IRB or Ethical Committee might want to ask you questions about being in the research, but you do not have to answer them. A court of law could order medical records shown to other people, but that is unlikely. Contacts: If you ever have any questions about the research study or study-related problems, you may contact Prof. Ben Gyan of the Noguchi Memorial Institute for Medical Research (0244 University of Ghana http://ugspace.ug.edu.gh 152 726016) at any time. For questions about the ethical aspects of this study or your rights as a volunteer, you may contact Dr. Chris Dadzie, Chairman, Institutional Review Board, NMIMR, University of Ghana (0302 501178/9) or Chairman of the Ghana Health Service Ethical Committee (Tel. 0302 681109) Your rights as a participant This research has been reviewed and approved by the NMIMR IRB and Ghana Health Service Ethical Committee. An IRB or Ethical Committee is a committee that reviews research studies in order to help protect participants. If you have any questions about your rights as a research participant you may contact Dr. Chris Dadzie, Tel 0302-501-178/179 or Chairman of the Ghana Health Service Ethical Committee (Tel. 0302 681109) VOLUNTEER AGREEMENT The above document describing the benefits, risks and procedures for the research title Circulating endothelial cells and the pathogenesis of malaria has been read and explained to me. I have been given an opportunity to have any questions about the research answered to my satisfaction. I agree my child/ward to participate as a volunteer. ------------------------------------------------ ------------------------------------------------ Date Signature or thumbprint of volunteer University of Ghana http://ugspace.ug.edu.gh 153 If volunteer’s Parent/Guardian cannot read the form themselves, a witness must sign here: I was present while the benefits, risks and procedures were read to the volunteer. All questions were answered and the volunteer’s Guardian/Parent has agreed to take part in the research. ------------------------------------------------ ---------------------------------------------- Date Signature or thumbprint of witness I certify that the nature and purpose, the potential benefits, and possible risks associated with participating in this research have been explained to the above individual. ------------------------------------------------ ----------------------------------------------------- Date Signature of person who obtained consent University of Ghana http://ugspace.ug.edu.gh 154 Appendix 3A: Assessment Forms/Questionnaires for Cerebral Malaria Cohort Endothelial progenitor cells and the pathogenesis of malaria CEREBRAL MALARIA COHORT INITIAL ASSESSMENT/QUESTIONNAIRE Initial Clinical Presentation I.1 Study ID number |__|__|__|__|__|__| I.2 Date of Admission ___/___/___ 1.2a Date of recruitment into study……..……………………………………__/___/___ I.3 Time of recruitment into study ___:_(AM or PM) I.4 Name………………………………_______________________________________ 1.5 Hospital name (circle) PML__Ridge__La General___ Tema General ___LEKMA I.5a Hospital Folder #...……………………………………………… |__|__|__|__|__|__| General Exclusion Criteria G.1 Pre-existing neurological disease (1=yes, 2 =no) |__| G.2 Recent severe head trauma (within 3 months)(1 = yes 2 = no) |__| G.3 Other cause of coma e.g. DM (1 = yes, 2 = no) |__| G.4 Blantyre Coma Score>3 (1=Yes, 2=No) ……………………………………… |__| G.5 Blantyre Coma Score >3 within 5 min of correction for hypoglycemia(1=Yes,2=No)|__| G.6 Duration of coma < 60 mins (1=Yes, 2=No) ……………………………………. |__| G.7 Other febrile illness (1=Yes, 2=No) …………………………………………… |__| G.8 Recent severe bleeding i.e. within 3 months (1=yes 2=no) |__| G.9 Other causes of anaemia including SCD and Sickle Cell trait (1= yes, 2=no) |__| G.10 Obvious clinical evidence of bacterial infection (1=yes, 2=no) |__| G.11 Obvious clinical evidence of viral infection (1=yes, 2=no) |__| G.12 History of Diabetes Mellitus (1=yes, 2=no) |__| G.13 History of cardiovascular disease (1=yes, 2=no) |__| G.14 History of Hypertension (1=yes, 2=no) |__| G.15 History of increased cholesterol (1=yes, 2=no)…………………… |__| G.16 History of surgery within 1 month? (1=yes, 2=no) |__| G.17 History of bone fracture within 3 months (1=yes, 2=no) |__| G.18 History chronic viral infection (chronic weight loss, diarrhea) (1=yes, 2=no) |__| NOTE –exclude HIV patients G.19 Major trauma (car accident, etc.) within 1 month (1=yes, 2=no) |__| G.20 Transfusion within past 3 months (1=Yes, 2=No) |__| G.21 Severe pallor (1=yes, 2=no)……………………………………………………… |__| If the answer to any of the above questions is yes (1), do not recruit into study! G.22 *****CONSENT REQUIRED****** please indicate obtained, 1=Yes 2=No......|__| Name of staff who obtained consent._______________________________ G.23 Ethnic origin |__| (Akan=1, Ga-Adangme=2, Ewe=3, Hausa=4, Frafra=5, Dagomba=6 University of Ghana http://ugspace.ug.edu.gh 155 Non-Ghanaian=7, Other=8 Specify _________________________ ) G.24 Area of residence/Direction to your house ****Cell phone #_____________ G.25 Sex (1=M, 2=F) |__| G.26 Age (Last half year passed) |__|__|.|__| G.27 Referral on the basis of a lab report positive for malaria parasites (1=Yes, 2=No) |__| G.28 History of a febrile illness in the preceeding 2 weeks (1=Yes, 2= No) |__| G.29 Duration of symptoms before presentation (Days same day = 1) |__| G.30 History of other antimalarial for this attack (1=yes, 2=no, 9=DK*) |__| G.31 If yes specify: …………. G.32 Reported cola urine (1=Yes, 2=No 9=DK ) |__| G.33 Observed cola urine (1=Yes, 2=No) |__| G.34 Reported convulsions (1=Yes, 2=No 9=DK) |__| G.35 Already seen at this hospital for this attack? (1=Yes, 2=No) |__| G.36 If yes initial antimalarial prescribed (……………………….) Physical exam, vital signs and laboratory results P.1 Best Motor Response (0-2) |__| P.2 Best Verbal Response (0-2) |__| P.3 Eye Movements (0-1) |__| P.4 Total Coma Score (0-5) |__| P.5 Duration of Coma (0=no coma, 1=0-60 mins, 2=60+ mins) |__| P.6. Observed Convulsions (1=Yes, 2=No) |__| P.7 Alar flare (1=Yes, 2=No) |__| P.8. Chest (subcostal, intercostal) Recession (1=Yes, 2=No) |__| P.9 Abnormally deep breathing (1=Yes, 2=No) |__| P.10 Use of Accessory muscles (supraclavicular/suprasternal recessions) (1=Yes, 2=No) |__| P.11 Fast breathing (1-4yr>40/min, >5yr>30/min) (1=yes, 2=no) |__| P.12 Respiratory Distress (1=Yes, 2=No)……………………………………………. |__| P.13 Peripheral O2 saturation (for all patients with resp. distress) |__|__| P.14 Temperature. |__|__|.|__| P.15 Weight (in kgs) |__|__|.|__| P.16 Height (in cms) |__|__|__| P.17 Blood Pressure (mmHg) |__|__|__|/|__|__| P.18 Pulse |__|__|__| P.19 State of hydration (1=normal, 2=impaired, i.e.,  skin turgor or dry mouth) |__| P.20 Recruited into study (1=Yes, 2=No) |__| Name:______________________________________________ Study id no. |__|__|__|__|__|__| Samples (Please tick when taken): University of Ghana http://ugspace.ug.edu.gh 156 S.1 ___ EDTApurple top (EPC sample) S.5 ___CXR ( for respiratory distress) S.2 ___ EDTA purple top (FBC-full blood count) S.6 ___CSF ( for CM) S.3 ____ Heparin tube(s) S.4 ____ Blood Culture S.5 _____PAX-gene tube (RNA) S.6 staff=nurse/MD filling out data ___________________________________________________ Results initial studies at recruitment: R.1aDate sample obtained (____/____/____) R.1 RBS: Glucometer ( mmol/L)..………………………………………… |__||__|.__| R.2 Haemoglobin (Hb). |__|__|.|__| R.3 WBC (X109/µL)…………………………………………………………… |__|__|.|__| RBC ____(X106/µL) Hemoglobin (Hb)_____(g/dL) HCT____ (%) MCV_______(fL) MCH______(pg) MCHC____(g/dL) PLT_____ (X103/µL) R.4 Blood film species (1=p.f.,2=p.m.,3=p.o., 4=p.v.,5=p.f.+p.m.,6=p.f.+p.o.) |__| R.5 Parasite density (per L)____________ R.6 Relative abundance (negative smear, 1+, 2+, 3+, 4+)……………………………|__| *Inform team if smear is negative and that repeat must be verified as positive within 48 hours for continued inclusion in the study. R.7 Asexual stage, density per l |__|__|__|__|__|__|__| R.8 Sickling status (1=positive, 2=negative) |__| NOTE- If positive then exclude the from study R.9 Blood culture results (1=positive, 2=negative |__| R.10 If positive organism cultured_________________________________________________ NOTE- If positive then exclude from the study University of Ghana http://ugspace.ug.edu.gh 157 Name:______________________________________________ Study id no. |__|__|__|__|__|__| Endothelial progenitor cells and the pathogenesis of malaria INPATIENT CM MONITORING CHART See footnote(s)/codes end of form Day0 *date_____ Day 1 *date_____ Day 2 *date_____ Day 3 *date_____ M.1 Asexual parasite count (per L) AM/date (If initial smear negative) 2 AM M.27 Coma score† M.28 Temp.oC M.29 BP M.30 Pulse/Resp Rate P___ /RR___ P___ /RR___ P___ /RR___ P___ /RR___ M.31 Staff name 6 AM M.2 Coma score† M.3 Temp. oC M.4 BP M.5 Pulse/Resp Rate P___ /RR___ P___ /RR___ P___ /RR___ P___ /RR___ M.6 Staff name 10 AM M.7 Coma score† M.8 Temp. oC M.9 BP M.10 Pulse/Resp Rate P___ /RR___ P___ /RR___ P___ /RR___ P___ /RR___ M.11 Staff name 2 PM M.12 Coma score† M.13 Temp.oC M.14 BP M.15 Pulse/Resp Rate P___ /RR___ P___ /RR___ P___ /RR___ P___ /RR___ M.16 Staff name 6 PM M.17 Coma score† M.18 Temp. oC M.19 BP University of Ghana http://ugspace.ug.edu.gh 158 M.20 Pulse/Resp Rate P___ /RR___ P___ /RR___ P___ /RR___ P___ /RR___ M.21 Staff name 10 PM M.22 Coma score† M.23 Temp oC M.24 BP M.25 Pulse/Resp Rate P___ /RR___ P___ /RR___ P___ /RR___ P___ /RR___ M.26 Staff name M.32Clinical developments 1=yes 2=no § use codes below, indicate time, name of staff making the entry, with additional information on notes page. 1=Y, 2=N____ code______ time________ staff________ 1=Y, 2=N____ code_________ time________ staff_________ 1=Y, 2=N__ code_______ time_______ staff_______ 1=Y, 2=N__ code_______ time_______ staff______ M.33 If change in status (recovery/worsening) indicate if blood samples taken (1=Y, 2=N) and check off which: FBC/EPC= purple top tubes Heparin tube(s) 1=Y, 2=N____ FBC/smear__ EPC_______ Heparin_____ Time samples obtained____ 1=Y, 2=N_____ FBC/smear____ EPC_________ Heparin_____ Time samples obtained______ 1=Y, 2=N__ FBC/smear_ EPC______ Heparin___ Time samples obtained____ 1=Y, 2=N__ FBC/smear_ EPC_______ Heparin____ Time samples obtained____  If initial blood smear and follow up smears are *negative* at day 2 (48hours) i.e. three consecutively negative smears then exclude patient from the study (assume patient does not have cerebral malaria – see protocol for meeting exclusion criteria (notify Study team, primary MD, etc.) †Blantyre score of 4 or 5 Blood samples to be obtained (EPC/FBC i.e. 2 purple top tubes AND heparin tube when patient recovers OR deteriorates University of Ghana http://ugspace.ug.edu.gh 159 Name:____________________________________ Study id no. |__|__|__|__|__|__| Endothelial progenitor cells and the pathogenesis of malaria CEREBRAL MALARIA COHORT HOSPITAL (INPATIENT) COURSE SUMMARY CS.1a Date of discharge*_____________CS.1b MD/RN completing summary________ Clinical Course (Refer to page prior pages for clinical notes/information) CS.1 Highest temperature first 24 hours |__|__|.|__| CS.2 Time for fever to settle (hrs) |___|___|___| (from admission till first time temp. falls to <37.5oC for at least 48 hours) CS.3 Time for coma score to reach 5 (hrs) |___|___|___| CS.4 Antimalarial changed before discharge? (1=Yes, 2=No)……. |__| CS.5 If yes; what was it changed to CS.6 Day of change (Day 0 = Admission day) |__| CS.7 Any other drugs (1=Yes, 2=No) |__| CS.8 If yes, specify ______________________________________________ CS.9 Blood C/S (1=positive‡, 2=negative, 3=not investigated, 9=missing) . |__| CS.10 If positive organism cultured _________________________ CS.11 Antibiotics prescribed (1=yes, 2=no)………………………. |__| CS.12 Name of antibiotic given_________________________ CS.13 CSF C/S (1=positive‡, 2=negative, 3=not investigated, 9=missing) . |__| CS.14 If positive organism cultured=_________________________ CS.15 Antibiotics prescribed (1=yes, 2=no)…………………………………………...|__| CS.16 Antibiotic given 1=ceftriaxone 2=other, give name____________________|__| CS.17 HB < 5 g/dL during admission (1=Yes, 2=No) |__| CS.18 Blood transfusion (1=Yes, 2=No) |__| CS.19 If yes day of transfusion (day 0=day of admission) |__| CS.20 Coca cola urine (1=Yes, 2=No) |__| CS.21 Respiratory distress (1=Yes, 2=No) |__| CS.22 Total number of convulsions |__| CS.23 Duration of longest convulsion (minutes) |__| CS.24 Died (1=Yes, 2=No) |__| CS.25 Interval between admission and death (hours) (10=NA) |__|__|__| CS.26 Postmortem done (1=Yes, 2=No) (10=NA) |__| CS.27 Additional / Differential Diagnoses _____________________________ Detailed information regarding address/contact_______________________________________ CS.28 Patient to continue in the study (1=Yes, 2=No)..……………………………….|__| CS.29 If excluded give reason ________________________________________________ CS.29 Scheduled days/times for return: CS.30 7 days post discharge=______________14 days post discharge=___________________ CS.31 Name MD who discharge patient/completed form_______________________________ University of Ghana http://ugspace.ug.edu.gh 160 Name:_________________________________ Study id no. |__|__|__|__|__|__| CEREBRAL MALARIA COHORT OUTPATIENT ASSESSMENT/QUESTIONAIRE **NOTE: EPC, FBC, parasite smear (2 purple tops) and 2 heparin tubes required for each follow up visit 7 DAYS POST RECOVERY Tick if visit is late ☐ QA1aDate_________ QA1b Staff_________ 14 DAYS POST RECOVERY Tick if visit is late☐ QA1a Date_________ QA1b Staff_________ HISTORY SINCE DISCHARGE OR LAST ASSESSMENT QA.1 Illness/change since discharge or last assessment (1=Yes, 2=No). QA.2 If yes; §use codes at end of form/describe if not listed, provide additional information on notes page or “10” if not applicable QA.3 Fever (1=Yes, 2=No) QA.4 Medical care/Hospitalization (1=Yes, 2=No) QA.5 If yes, was the medical care or hospitalization malaria related 1=Yes, 2=No QA.6 Convulsion or impaired consciousness (1=Yes, 2=No) QA.7 Severe bleeding (1=Yes, 2=No) QA.8 Trauma (1=Yes, 2=No) QA.9 If yes, indicate type (eg car accident)_________________ QA.10 Surgery (1=Yes, 2=No) QA.11 If yes, indicate type_______ QA.12 New antibiotics taken (1=Yes, 2=No) QA.13 If yes specifiy;__________ ___ QA.14 New antimalarial taken (1=Yes, 2=No) CLINICAL ASSESSMENT Q15. Temperature oC Q16. BP (mm Hg) Q17. Pulse University of Ghana http://ugspace.ug.edu.gh 161 Q19. Weight (in kgs) Q19aHeight (in cms) Q20. Neurologic sequelae at last assessment (1=Yes, 2=No) Q21. Resolution of neurologic sequelae since last assessment (1=Yes, 2=No, 10=not applicable) Q22. If yes indicate deficit type*, if other specify;________________________ Q23. If no, indicate if deficit has improved (1=Yes, 2=No, 3=not applicable) Q24. Describe improvement______________ Q25. Blood sample obtained (1=Y, 2=N). Check off which: FBC and EPC= purple top tubes (1 each) 1=Y, 2=N____ FBC/smear__ EPC_____ Heparin___ 1=Y, 2=N____ FBC/smear__ EPC_____ Heparin___ Q26. Parasite density (per microlitres) Asexual stage, density per l Q27. WBC X109/L Q28. HB (g/dL) Q29. Platelet count University of Ghana http://ugspace.ug.edu.gh 162 Appendix 3B: Assessment Forms/Questionnaires for Uncomplicated Malaria Cohort Endothelial progenitor cells and the pathogenesis of malaria UNCOMPLICATED MALARIA COHORT INITIAL ASSESSMENT/QUESTIONNAIRE Initial Clinical Presentation IU.1 Study ID number.............................................................................. |__|__|__|__|__|__| IU.2 Date of assessment .................................................................................... ___/___/___ IU.3 Time of assessment ..................................................................... ___:___hrs AM/PM IU.4 Name. ..................................................._____________________________________ IU.5 Hospital name/ (circle)PML____Ridge___La General__TemaGeneral___LEKMA___ IU.6 Hospital Folder #...............................................................................|__|__|__|__|__|__| General Exclusion Criteria GU.1 Blantyre Coma Score<5 (1=Yes, 2=No) ……………………………………… |__| GU.2 Blood film negative for P. falciparum asexual forms (1=Yes, 2=No) ……….. |__| GU.3 Recent severe bleeding i.e. within 3 months (1=yes 2=no) ................................ |__| GU.4 Other causes of anaemia including SCD and Sickle Cell trait (1= yes, 2=no) ... |__| GU.5 Obvious clinical evidence of bacterial infection (1=yes, 2=no) ......................... |__| GU.6 Obvious clinical evidence of viral infection (1=yes, 2=no) ................................ |__| GU.7 History of Diabetes Mellitus (1=yes, 2=no) ......................................................... |__| GU.8 History of cardiovascular disease (1=yes, 2=no) ................................................. |__| GU.9 History of Hypertension (1=yes, 2=no) ............................................................... |__| GU.10 History of increased cholesterol (1=yes, 2=no)…………………… ................... |__| GU.11 History of surgery within 1 month? (1=yes, 2=no) .............................................. |__| GU.12 History of bone fracture within 3 months (1=yes, 2=no) ..................................... |__| GU.13 History chronic viral infection (chronic weight loss, diarrhea) (1=yes, 2=no) .... |__| NOTE –exclude HIV patients GU.14 Major trauma (car accident, etc.) within 1 month (1=yes 2=no) ........................ |__| GU.15 Transfusion within past 3 months (1=Yes, 2=No) .............................................. |__| GU.16 Moderate or Severe pallor (1=yes, 2=no)………………………………………|__| GU.17 Reported Convulsions (1=yes, 2=no) ................................................................. |__| GU.18 Reported Cola urine (1=yes, 2=no) ..................................................................... |__| GU.19 Respiratory distress (1=yes, 2=no) ...................................................................... |__| GU.20 Requires hospitalization (1=yes, 2=no) .............................................................. |__| If the answer to any of the above questions is yes, do not recruit into study! GU.21 *****CONSENT REQUIRED****** please indicate obtained, 1=Yes 2=No ..|__| Name of staff who obtained consent._______________________ GU.22 Ethnic origin ........................................................................................................ |__| University of Ghana http://ugspace.ug.edu.gh 163 (Akan=1, Ga-Adangme=2, Ewe=3, Hausa=4, Frafra=5, Dagomba=6 Non-Ghanaian=7, Other=8 Specify _________________________ ) GU.23 Area of residence/Direction to house (eg Any popular Spot or street etc) _________________________________________________ Cell phone #____________ GU.24 Sex (1=M, 2=F) .................................................................................................. |__| GU.25 Age (Last half year passed) .....................................................................|__|__|.|__| GU.26 Referral on the basis of a lab report positive for malaria parasites (1=Yes, 2=No)|__| GU.27 History of a febrile illness in the preceding 2 weeks (1=Yes, 2= No) ................ |__| GU.28 Duration of symptoms before presentation (Days, same day = 1) ...................... |__| GU.29 History of other antimalarial for this attack (1=yes, 2=no, 9=DK) .................... |__| GU.30 If yes specify: GU.31 Observed cola urine (1=Yes, 2=No) ................................................................... |__| NOTE- If cola urine=Yes (1) do not recruit into the study! Physical Exam, Vital Signs and Laboratory Results PU.1 Temperature (At time of blood collection). .............................................|__|__|.|__| PU.2 Weight (in kgs) .......................................................................................|__|__|.|__| PU.3 Height (in cms) .......................................................................................... |__|__|__| PU.4 Blood Pressure (mmHg) ................................................................ |__|__|__|/|__|__| PU.5 Pulse ........................................................................................................... |__|__|__| PU.6 Best Motor Response (0-2) .................................................................................. |__| PU.7 Best Verbal Response (0-2) ................................................................................. |__| PU.8 Eye Movements (0-1) .......................................................................................... |__| PU.9 Total Coma Score (0-5) ...................................................................................... |__| NOTE – if < 5 notify study principles for possible inclusion in CM study group- Do not enroll in UM if<5 PU.10 Alar flare (1=Yes, 2=No) .................................................................................... |__| PU.11 Chest (subcostal, intercostal) Recession (1=Yes, 2=No) .................................... |__| PU.12 Abnormally deep breathing (1=Yes, 2=No) ....................................................... |__| PU.13 Use of Accessory muscles (supraclavicular/suprasternal recessions) (1=Yes, 2=No) .......................................................................................................................................... |__| PU.14 Fast breathing (1-4yr>40/min, >5yr>30/min) (1=yes, 2=no) ................................... PU.15 Respiratory Distress (1=Yes, 2=No). .................................................................. |__| PU.16 State of hydration (1=normal, 2=impaired, ie skin turgor or dry mouth) ........ |__| PU.17 Spleen size (cm below costal margin). .......................................................... |__|__| PU.18 Liver size (cm below costal margin)… .......................................................... |__|__| PU.19 Antimalarial (you) prescribed (i.e., received by patient today)……….… |__| Samples (Please tick when taken): SU.1 ___ EDTA purple top(EPC) _____ SU.5 _____PAX-gene tube (RNA) SU.2 ___ EDTA purple top (FBC/CBC) SU.3 ___ Blood C/S SU.4 __ Heparin tube(s) University of Ghana http://ugspace.ug.edu.gh 164 Results day of recruitment : RU.1 Haemoglobin (Hb). ....................................................................................|__|__|.|__| RU.2 WBC (X109/L)..…………………………………………………………|__|__|.|__| RBC ____(X106/µL) Hemoglobin (Hb)____(g/dL) HCT____ (%) MCV____(fL) MCH______(pg) MCHC____(g/dL) PLT_____ (X103/µL) RU.13 Platelet count………………………………………………………___________ RU.14 Blood Film species (1=p.f.,2=p.m.,3=p.o., 4=p.v.,5=p.f.+p.m.,6=p.f.+p.o.) . |__| RU.15 Parasite Density (per microlitres) (NOTE- if <2500/l then exclude from the study) RU.16 Asexual stage, density per l ................................................ |__|__|__|__|__|__|__| RU.17 Sickling status (1=positive, 2=negative) ............................................................ |__| (NOTE- If positive then exclude from the study) RU.18 Blood culture results (1=positive, 2=negative) .......................................... |__| RU.19 If positive organism cultured___________________________________________ NOTE- If positive then exclude from the study University of Ghana http://ugspace.ug.edu.gh 165 Name:__________________________________ Study id no. |__|__|__|__|__|__| Endothelial progenitor cells and the pathogenesis of malaria UNCOMPLICATED MALARIA COHORT FOLLOW-UP ASSESSMENT/QUESTIONNAIRE **NOTE: FBC, parasite smear & EPC (2 purple tops) and heparin tube(s)required for each follow up visit 7 DAYS POST RECRUITMENT 14 DAYS POST RECRUITMENT HISTORY SINCE DISCHARGE OR LAST ASSESSMENT Tickif visit is late☐ OU.1aDate_______ OU.1b Staff________ Tick if visit is late☐ OU.1a Date______ OU.1b Staff_______ OU.1 Illness/change since discharge or last assessment (1=Yes, 2=No). OU.2 If yes; §use codes at end of form/describe if not listed, provide additional information on notes page OU.3 Fever (1=Yes, 2=No) OU.4 Medical care/Hospitalization (1=Yes, 2=No) OU.5 If yes, was the medical care or hospitalization malaria related 1=Yes, 2=No) OU.6 Convulsion or impaired consciousness (1=Yes, 2=No) OU.7 Severe bleeding (1=Yes, 2=No) OU.8 Trauma (1=Yes, 2=No) OU.9 If yes, indicate type (eg car accident)_______________ OU.10 Surgery (1=Yes, 2=No) OU.11 If yes, indicate type_________________ OU.12 New antibiotics taken (1=Yes, 2=No) OU.13 If yes specifiy;___________________ OU.14 New antimalarial taken (1=Yes, 2=No) OU.15 If yes, specify_____________________ CLINICAL ASSESSMENT OU.16 Temperature oC University of Ghana http://ugspace.ug.edu.gh 166 OU.17 BP (mm Hg) OU.18. Pulse OU.9. Weight (in kgs) OU.20 Neurologic sequelae at last assessment (1=Yes, 2=No) OU 21 Resolution of neurologic sequelae since last assessment (1=Yes, 2=No, 10=not applicable) OU.22 If yes indicate deficit type*……………………….. OU.23 If no, indicate if deficit has improved (1=Yes, 2=No, 3=not applicable) OU.24 Describe improvement______________ OU.25. Blood sample obtained (1=Y, 2=N). Check off which: FBC and EPC= purple top tubes (1 each) Heparin=red top tube 1=Y, 2=N_______ FBC/smear_____ EPC__________ Heparin________ 1=Y, 2=N_______ FBC/smear_____ EPC__________ Heparin________ OU.26 Parasite density (per microlitres) Asexual stage, density per l OU.27 WBC X109/L OU.28 HB (g/dL) OU.29 Platelet count §Indicate all changes in clinical status University of Ghana http://ugspace.ug.edu.gh 167 Appendix 3C: Assessment Form/Questionnaire for Healthy Volunteer Cohort Endothelial progenitor cells and the pathogenesis of malaria HEALTHY CONTROLS INITIAL STUDY QUESTIONNAIRE IHC.1 Location of Recruitment_____________________________________________ OR IHC.2 Hospital Folder #....................................................................................|__|__|__|__|__|__| IHC.3 Date of recruitment……………………………………………………….___/___/____ IHC.4 Time of recruitment………………………………………………_____:_____AM/PM General Exclusion Criteria GC.1 Recent severe bleeding e.g. within 3 months (1=yes, 2=no) ................................. |__| GC.2 Other causes of anaemia including SCD and Sickle Cell trait (1= yes, 2=no) ..... |__| GC.3 Obvious clinical evidence of bacterial infection (1=yes, 2=no) ............................ |__| GC.4 Obvious clinical evidence of viral infection (1=yes, 2=no) .................................. |__| GC.5 History of diabetes mellitus (1=yes, 2=no) ........................................................... |__| GC.6 History of cardiovascular disease (1=yes, 2=no) .................................................. |__| GC.7 History of hypertension (1=yes, 2=no) .................................................................. |__| GC.8 History of increased cholesterol (1=yes, 2=no)…………………… .................... |__| GC.9 History of surgery within 1 month? (1=yes, 2=no) ................................................ |__| GC.10 History of fever within last 2 weeks (1=yes, 2=no) ............................................. |__| GC.11 History of treatment for malaria within the last two weeks(1=yes, 2=no) ........... |__| GC.12 History of cola urine within the last 2 weeks (1=yes, 2=no) ................................ |__| GC.13 History of history convulsions within the last 2 weeks (1=yes, 2=no) ................ |__| GC.14 History of bone fracture within 3 months (1=yes, 2=no) ..................................... |__| GC.15 History chronic viral infection (chronic weight loss, diarrhea) (1=yes, 2=no) .... |__| NOTE – exclude HIV patients GC.16 Major trauma (car accident, etc.) within 1 month (1=yes, 2=no) ......................... |__| GC.17 Transfusion within past 3 months (1=yes, 2=no) ................................................. |__| Severe pallor (1=yes, 2=no)………………………………………………………….. |__| If the answer to any of the above questions is yes, do not recruit into study! GC.18 *****CONSENT REQUIRED****** please indicate obtained, 1=Yes…………………… |___| Name of staff who obtained consent._________________________________________ GC 18b. Name of medical professional taking history/physical_________________________ University of Ghana http://ugspace.ug.edu.gh 168 Name:___________________________________________________ ID no. |__|__|__|__|__|__| GC.19 Area of residence:______________________________________________________ ______________________________________________Cell phone #__________________ GC.20 Ethnic origin ......................................................................................................... |__| (Akan=1, Ga-Adangme=2, Ewe=3, Hausa=4, Frafra=5, Dagomba=6 Non-Ghanaian=7, Other=8 Specify _________________________ ) GC.21 Sex (1=M, 2=F) ................................................................................................... |__| GC.22 Age (Last half year passed) ......................................................................|__|__|.|__| GC.23 Temperature ..............................................................................................|__|__|.|__| GC.24 Blood Pressure (mmHg) ................................................................. |__|__|__|/|__|__| GC.25 Pulse ........................................................................................................... |__|__|__| GC.26 Weight (in kgs) ........................................................................................|__|__|.|__| GC.26a Height (in cm) ........................................................................................ |__|__|__| GC. 27 Samples (Please tick when taken): ___ EDTA purple top (EPC sample) _____PAX-gene tube (RNA) ___ EDTA purple top (FBC/CBC-full blood count) ___ Heparin tube Results on day of recruitment: GC.28 Hemoglobin (Hb). |__|__|.|__| GC.29 WBC (X109/L)………|__|__|.|__| GC.30 RBC_________________(x106/μL) GC.30a Hb__________________ (g/dL) GC.31 HCT_________________ (%) GC.32 MCV_________________ (fL) GC.32a MCH_________________(pg) GC.32b MCHC________________(g/dL) GC.33 PLT__________ ________(x103/μL) GC.34 LYMPH%______________(%) GC.35 MXD%________________(%) GC.38 NEUT%_______________(%) GC.38a LYM#_________________(x103/μL) GC.38b MXD#_________________(x103/μL) GC.38c NEUT#________________(x103/μL) GC.39 RDW(SD) _____________(fL) GC.39a RDW-CV______________(%) GC.40 PDW_________________(fL) GC.40a MPV_________________(fL) GC.40b P-LCR________________(%) GC.41 Blood Film species (1=p.f.,2=p.m.,3=p.o., 4=p.v.,5=p.f.+p.m.,6=p.f.+p.o.) ... |__| University of Ghana http://ugspace.ug.edu.gh 169 GC.42 Parasite density (per L) GC.43 Asexual stage, density per l .................................................. |__|__|__|__|__|__|__| GC.44 Sickling status (1=positive, 2=negative) .............................................................. |__| NOTE- if positive then exclude from the study HEALTHY CONTROLS FOLLOW-UP ASSESSMENT/QUESTIONNAIRE 7 DAYS POST INITIAL ASSESSMENT 14 DAYS POST INITIAL ASSESSMENT HISTORY SINCE LAST ASSESSMENT* Date_________ Date________ QC.1 Illness since last assessment (1=Yes, 2=No) QC.2 If yes; describe (diarrhea, cough, infection etc) QC.3 Fever (1=Yes, 2=No) QC.4 Medical care/Hospitalization (1=Yes, 2=No) QC.5 If yes, was the medical care or hospitalization malaria related(1=Yes, 2=No) QC.6 Severe bleeding (1=Yes, 2=No) QC.7 Convulsion or impaired consciousness (1=Yes, 2=No) QC.8 Trauma (1=Yes, 2=No) QC.9 If yes, indicate type (eg car accident)_________________ QC.10 Surgery (1=Yes, 2=No) QC.11 If yes, indicate ___________________________________ QC.12 Antibiotics (1=Yes, 2=No) QC.13 If yes specifiy;____________________________________ QC.14 Antimalarials (1=Yes, 2=No) QC.15 If yes‡, specify; CLINICAL ASSESSMENT QC.18 Temperature oC, QC.19 BP (mm Hg) University of Ghana http://ugspace.ug.edu.gh 170 QC.20 pulse QC.21 Weight (in kgs) QC.22 Blood samples obtained (indicate test EPC, FBC, heparin: 1=Yes, 2=No, 3=ND – if not state why) QC.23 Parasite density (per microlitres) QC.24 Asexual stage, density per l QC.25 WBC X109/L QC.28 HB (g/dL) *‡IF PATIENT REFERRED FOR MEDICAL CARE DUE TO MALARIA ALERT for possible inclusion in malaria arm but cannot be included as a healthy control. 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