University of Khartoum Graduate College Medical and Health Studies Board
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University of Khartoum Graduate College Medical and Health Studies Board Pathophysiology of Severe Malaria in Children: Clinical and Laboratory Correlations A thesis submitted for the degree of Ph. D in Human Physiology By Nisreen Daffalla Omer Haj Edris M.B.B.S Supervisor Professor Mohammed Yousif Sukkar M.B.B.S, Ph. D in Human Physiology 2010 Table of contents Dedication I Acknowledgements II Abbreviations III Abstract IV-VI Arabic abstract VII-IX List of figures X List of tables XI CHAPTER ONE: INTRODUCTION & LITERATURE REVIEW & OBJECTIVES OF THE STUDY Introduction 1-3 Malaria parasite life cycle 4 Clinical presentation of malaria 5 Uncomplicated malaria 6 Severe malaria 6 Pathophysiology of severe malaria 7 Overview of immunity and cytokines in malaria 7 Cytokines members: Interleukins 8-9 IL-10 10 Chemokines 11 Interferons 12 Tumor necrosis factor-alpha 12-14 General properties of cytokines receptors 14 Effect of cytokines on the immune system 14-15 Cytokines and immunity in malaria 15-17 Innate immunity 17-19 The role of nitric oxide in malaria 19-20 Adaptive immunity 20-21 Cytokines in the immunopathology of malaria 22 Serious complications of malaria 23 Cerebral malaria 23-29 Repeated convulsions 29 Severe anaemia 30-34 Hypoglycemia 34-37 Renal involvement in malaria 37-39 Electrolyte changes 39-41 Haemoglobinuria 42-43 Objectives 44 CHAPTER TWO: PATIENTS AND METHODS Location and time of the study 45 Patients and control 45 History and physical examination 45 Malaria diagnosis and blood tests 45 Blood samples 45 Malaria diagnosis and parasitemia estimation 46 Haemoglobin estimation 46 Total white blood cells count 47 Random blood glucose measurement 47 Hormone assays; insulin and C-peptide measurement 47-49 Urea measurement 49 Creatinine measurement 49 Sodium and potassium measurement 50 Bilirubin measurement 50 Alanine aminotransferase measurement 51 Aspartate aminotransferase measurement 52 Alkaline phosphatase measurement 52 Cytokines measurement 53 IFN-γ assay 53 TNF-α assay 54 IL-10 assay 54 Stastical methods 55 CHAPTER THREE: RESULTS Sample characteristics 56 Clinical presentation 57 Blood glucose 58 Insulin 59 C- peptide 60 Insulin: C-peptide ratio 61 Correlation between insulin and blood glucose 62 Correlation between insulin and C-peptide 63 Haemoglobin 64 Total white blood cells count 65 Bilirubin 66 Liver enzymes 67-70 Urea 70 Creatinine 71 Sodium 72-73 Potassium 74 Association of the course of malaria and the clinical categories 75 Mixed presentation 75 Cerebral malaria 76 Parasitemia 76-79 Period of illness 79 TNF-α 80-82 IFN-γ 83-87 IL-10 87-89 Correlation of cytokines with age 90-91 CHAPTER FOUR: DISCUSSION Introduction 92 Effect of age on the severity of malaria 92-93 Anaemia in severe malaria 94-96 Liver involvement in severe malaria 96-99 Spleen in severe malaria 99-101 TWBCs 101 Blood glucose homeostasis 102-106 Insulin and C-peptide 106-101 Disturbances of electrolytes and renal functions 107-111 Sodium 107 Potassium 108 Urea 109 Creatinine 110 Haemoglobinuria 111 Cytokines in severe malaria 112-114 TNF-α 114-117 IL-10 117-121 IFN-γ 121-125 Genetic profile 125 Cerebral malaria 126-129 Repeated convulsions 130 Indicators of poor outcome 131-134 Conclusions 135-139 Recommendations 140 References 141-188 Appendix 189-191 Dedication I dedicate this work to my parents, my sisters, my husband and to my children Mohammed and Abdelrahman. I Acknowledgments I wish to express my deep gratitude and special thanks to Professor Mohammed Yousif Sukkar, Department of Physiology, Faculty of Medicine, University of Khartoum and Nile College, for his unlimited supervision, encouragement and moral support. His scholastic approach, clear conception and insight of the problem have opened a new vista which profoundly enlightened my knowledge and helped me to understand the complicated issues. Also he has been very kind in checking the script of the thesis, Iam very grateful to Dr. Abdelsalam Alnoor, Dr.Amal Saeed and Professor. Ammar Eltahir, Department of Physiology, Faculty of Medicine, University of Khartoum for their unlimited support and encouragement. I would like to thank those volunteers who provided blood samples for the study. I am obliged to Wadmedani Paediatric Hospital main laboratory staff for their cooperation and their help with the laboratory analysis. My special thanks go to the International laboratory staff for their kind support and help with the cytokines measurement. I wish to thank Adil Amin for his help with the stastical analysis. My sincere thanks are also due to my parents, sisters, brothers and my husband for their patience, support and continued encouragement which kept the momentum of my research going. II Abbreviations SM: Severe malaria UM: Uncomplicated malaria CM: Cerebral malaria SA: Severe anaemia NO: Nitric oxide RBCs: Red blood cells Hb: Haemoglobin RBG: Random blood glucose I: C: Insulin C-peptide ratio TWBCs: Total white blood cells III ABSTRACT Background Despite the programmes of malaria control the incidence of mortality from severe malaria (SM) continues to rise. This is mainly attributable to the fact that the pathophysiological mechanisms are still not clearly defined. Objectives This project investigated the endocrine and paracrine aspects of the pathophysiology of SM in children. The study monitored relevant clinical and laboratory parameters of SM cases on admission so as to identify and correct life threatening changes. Patients and Methods This is a cross sectional study conducted at Wadmedani Paediatric Hospital from September 2007 to September 2008. Fifty six Children with SM, identified according to WHO criteria, were included in the study. Thirty one children with uncomplicated malaria (UM) were included for comparison. Both groups underwent a history and physical examination. Investigations included: parasitemia, Hb, RBG, TWBC, liver enzymes, bilirubin, urea, creatinine, sodium, potassium and cytokines (TNF-α, IFN-γ, IL-10). Statistical analysis was carried out with SPSS for windows. Means were analyzed by student’s T test. Correlations were analyzed using χ2 tests, Pearson's correlation coefficient and Spearman' rank correlation coefficient tests. Assessments of prognostic factors were conducted with regression model. P values < 0.05 were considered significant. Results Children with SM were younger than those with UM (mean age=56.75 months compared to 82.71 months, P value= 0.001). IV A significant difference was found in the mean RBG between children with SM and UM (89.27±39.06 compared to 108.06±39.14 mg/dl respectively) P value= 0.035 as well as in the mean Hb (7.54±2.71 compared to 9.91±1.67 mg/dl respectively, P value= 0.000). The pattern and cytokines secretion levels showed higher mean TNF-α in children below 5 years old and in children with CM. Those above 5 years had higher IFN-γ which was found to correlate positively with Hb, RBG, splenomegaly and hepatomegaly. Children with SA had a lower IFN-γ and IL-10 when compared to UM. Conclusions SA may result from haemolysis of red blood cells, low level of IFN-γ and IL-10. Glucose consumption by the parasites and low IFN-γ may be the causes of hypoglycemia. CM, mixed presentation, young age and hyperkalaemia were independently associated with increased risk of morbidity and mortality. High bilirubin, high TWBCs counts, high urea, high creatinine, low Hb and low RBG were significantly associated with CM and mixed presentation. V ﻣﻠﺨﺺ اﻟﺪراﺳﺔ ﺧﻠﻔﻴﺔ ﻋﻠﻲ اﻟﺮﻏﻢ ﻣﻦ ﺑﺮاﻣﺞ ﻣﻜﺎﻓﺤﺔ اﻟﻤﻼرﻳﺎ اﻻ ان ﻣﻌﺪل اﻟﻮﻓﻴﺎت ﻣﺎزاﻟﺖ ﻓﻲ ارﺗﻔﺎع ﻣﺴﺘﻤﺮ. وهﺬا ﻳﻌﺰي اﺳﺎﺳﺎ اﻟﻲ ﺣﻘﻴﻘﺔ ان اَﻟﻴﺎت اﻟﻔﺴﻴﻮﻟﻮﺟﻴﺎ اﻟﻤﺮﺿﻴﺔ ﻻﺗﺰال ﻏﻴﺮ ﻣﻌﺮوﻓﺔ اﻻهﺪاف هﺬﻩ اﻟﺪراﺳﺔ ﺗﺒﺤﺚ ﻓﻲ اﻟﻔﺴﻴﻮﻟﻮﺟﻴﺎ اﻟﻤﺮﺿﻴﺔ ﻟﻠﻤﻼرﻳﺎ اﻟﻮﺧﻴﻤﺔ. ﺗﺮﺻﺪ هﺬﻩ اﻟﺪراﺳﺔ اﻟﻤﺆﺷﺮات اﻟﺴﺮﻳﺮﻳﺔ واﻟﻤﻌﻠﻤﻴﺔ ﺧﻼل اﻟﻤﺮاﺣﻞ اﻟﻤﺒﻜﺮة ﻟﻠﻤﺮض وذﻟﻚ ﻟﺘﺤﺪﻳﺪ وﺗﺼﺤﻴﺢ اﻟﺘﻐﻴﻴﺮات اﻟﺘﻲ ﺗﻬﺪد اﻟﺤﻴﺎة. اﻟﻤﺮﺿﻲ واﻟﻮﺳﺎﺋﻞ: ﻳﻘﻮم هﺬا اﻟﺒﺤﺚ ﻋﻠﻲ دراﺳﺔ ﻣﻘﻄﻌﻴﺔ ﻋﻠﻲ ﻣﺠﻤﻮﻋﺔ ﻣﻦ اﻟﻤﺮﺿﻲ. اﺟﺮﻳﺖ هﺬﻩ اﻟﺪراﺳﺔ ﻓﻲ ﻣﺴﺘﺸﻔﻲ ودﻣﺪﻧﻲ ﻟﻼﻃﻔﺎل ﻓﻲ اﻟﻔﺘﺮة ﻣﻦ ﺳﺒﺘﻤﺒﺮ 2007 ﺣﺘﻲ ﺳﺒﺘﻤﺒﺮ2008. ﺷﻤﻠﺖ اﻟﺪراﺳﺔ 56 ﻃﻔﻞ ﻳﻌﺎﻧﻮن ﻣﻦ اﻟﻤﻼرﻳﺎ اﻟﻮﺧﻴﻤﺔ وﻗﺪ ﺗﻢ ﺗﺤﺪﻳﺪهﻢ وﻓﻘﺎ ﻟﻤﻌﺎﻳﻴﺮ ﻣﻨﻈﻤﺔ اﻟﺼﺤﺔ اﻟﻌﺎﻟﻤﻴﺔ. ﺷﻤﻠﺖ اﻟﺪراﺳﻪ ا ﻳ ﻀ ﺎ31ُ ﻃﻔﻞ ﻣﻦ اﻟﺬﻳﻦ ﻳﻌﺎﻧﻮن ﻣﻦ اﻟﻤﻼرﻳﺎ ﻏﻴﺮ اﻟﻮﺧﻴﻤﺔ آﻠﺘﺎ اﻟﻤﺠﻤﻮﻋﺘﻴﻦ ﺧﻀﻌﺖ ﻟﻠﻔﺤﺺ اﻟﺴﺮﻳﺮي واﻻﺧﺘﺒﺎرات اﻟﻤﻌﻤﻠﻴﻪ اﻟﺘﺎﻟﻴﺔ: ﻣﻌﺪل ﺗﻮاﺟﺪ اﻟﻄﻔﻴﻞ، اﻟﻬﻴﻤﻮﻏﻠﻮﺑﻴﻦ، ﻋﺪد آﺮﻳﺎت اﻟﺪم اﻟﺒﻴﻀﺎء ، اﻧﺰﻳﻤﺎت اﻟﻜﺒﺪ، اﻟﺒﻴﻠﻮروﺑﻴﻦ ، اﻟﺒﻮﻟﻴﻨﺎ ، اﻟﻜﺮﻳﺎﺗﻴﻨﻴﻦ ، اﻟﺼﻮدﻳﻢ ، اﻟﺒﻮﺗﺎﺳﻴﻮم و اﻟﺴﺎﻳﺘﻮآﺎﻳﻨﺎت اﻻﺗﻴﻪ (TNF-α, IFN-γ, IL-10). اﺳﺘﺨﺪم SPSS ﻟﻠﺘﺤﻠﻴﻞ اﻻﺣﺼﺎﺋﻰ. ﻗﻮرﻧﺖ اﻟﻤﺘﻮﺳﻄﺎت ﺑﺎﺧﺘﺒﺎر student’s T test. ﻟﺘﺤﻠﻴﻞ اﻻرﺗﺒﺎﻃﺎت اﺳﺘﺨﺪﻣﺖ اﻻﺧﺘﺒﺎرات اﻻﺗﻴﻪχ2 tests و Pearson's correlation coefficient and Spearman' rank correlation coefficient tests. ﺗﻘﻴﻴﻢ اﻟﻌﻮاﻣﻞ اﻟﺘﻨﺒﺆﻳﻪ اﺟﺮى ﺑﻮاﺳﻄﺔ regression model. ﻗﻴﻤﺔ P اﻗﻞ ﻣﻦ 0.05 اﻋﺘﺒﺮت ﻓﺮق ﻣﻌﻨﻮى. اﻟﻨﺘﺎﺋﺞ: اﻻﻃﻔﺎل اﻟﻤﺼﺎﺑﻮن ﺑﺎﻟﻤﻼرﻳﺎ اﻟﻮﺧﻴﻤﺔ آﺎﻧﻮا ﻣﻦ ذوي اﻻﻋﻤﺎر اﻟﺼﻐﻴﺮة ﻣﻘﺎرﻧﺔ ﺑﻤﺮﺿﻲ اﻟﻤﻼرﻳﺎ ﻏﻴﺮ اﻟﻮﺧﻴﻤﺔ (ﻣﺘﻮﺳﻂ اﻟﻌﻤﺮ75.56 ﺷﻬﺮﻣﻘﺎرﻧﻪ 82.71 ﺷﻬﺮ) ﻗﻴﻤﺔ P=0.001 وﺟﺪ ﻓﺮق ﻣﻌﻨﻮي ﻓﻲ ﻣﺴﺘﻮي اﻟﺠﻠﻜﻮز ﻓﻲ اﻟﺪم ﺑﻴﻦ ﻣﺮﺿﻲ اﻟﻤﻼرﻳﺎ اﻟﻮﺧﻴﻤﺔ وﻏﻴﺮ اﻟﻮﺧﻴﻤﺔ (89.27±39.06 - 108.6±39.14 ﻣﻠﺠﻢ/ دﺳﻠﺘﺮ ﻋﻠﻲ اﻟﺘﻮاﻟﻲ) ﻗﻴﻤﺔ P=0.035 آﺬاﻟﻚ ﻓﻰ اﻟﻬﻴﻤﻮﻏﻠﻮﺑﻴﻦ (7.54±2.71ﻣﻠﺠﻢ/ دﺳﻠﺘﺮ -9.91±1.67 ﻣﻠﺠﻢ/ دﺳﻠﺘﺮ ﻋﻠﻲ اﻟﺘﻮاﻟﻲ) ﻗﻴﻤﺔ P=0.000. VI اﻻﻃﻔﺎل دون ﺳﻦ اﻟﺨﺎﻣﺴﺔ ارﺗﻔﻊ ﻟﺪﻳﻬﻢ TNF-α آﻤﺎ ارﺗﻔﻊ ﻋﻨﺪ ﻣﺮﺿﻲ اﻟﻤﻼرﻳﺎ اﻟﺪﻣﺎﻏﻴﺔ. اﻻﻃﻔﺎل ﻓﻮق ﺳﻦ اﻟﺨﺎﻣﺴﺔ ارﺗﻔﻊ ﻟﺪﻳﻬﻢ IFN-γ اﻟﺬى وﺟﺪ اﻧﻪ ﻳﺮﺗﺒﻂ ارﺗﺒﺎط اﻳﺠﺎﺑﻲ ﻣﻊ اﻟﻬﻴﻤﻮﻏﻠﻮﺑﻴﻦ ، اﻟﺠﻠﻜﻮز، ﺗﻀﺨﻢ اﻟﻄﻮﺣﺎل وﺗﻀﺨﻢ اﻟﻜﺒﺪ. ﻣﺮﺿﻲ ﻓﻘﺮ اﻟﺪم اﻟﺤﺎد ﻧﻘﺺ ﻋﻨﺪهﻢ ﻣﺴﺘﻮي IFN-γ و-IL 10 اآﺜﺮ ﻋﻨﺪ اﻟﻤﻘﺎرﻧﺔ ﺑﻤﺮﺿﻲ اﻟﻤﻼرﻳﺎ ﻏﻴﺮ اﻟﻮﺧﻴﻤﺔ و اﻟﺬﻳﻦ ﻻﻳﻌﺎﻧﻮن ﻣﻦ ﻓﻘﺮ اﻟﺪم اﻟﺤﺎد. اﻟﺨﻼﺻﻪ ﻓﻘﺮ اﻟﺪم اﻟﺤﺎد ﻳﻨﺘﺞ ﻋﻦ ﺗﻜﺴﺮ آﺮﻳﺎت اﻟﺪم اﻟﺤﻤﺮاء، ﺗﺜﺒﻴﻂ ﻋﻤﻠﻴﺔ ﺗﺼﻨﻴﻊ آﺮﻳﺎت اﻟﺪم اﻟﺤﻤﺮاء وﻧﻘﺺ ﻣﺴﺘﻮى IFN-γ و IL-10 . إﻧﺨﻔﺎض اﻟﺠﻠﻜﻮز اﻟﺤﺎد رﺑﻤﺎ ﻳﻨﺘﺞ ﻋﻦ إﺳﺘﻬﻼك اﻟﻄﻔﻴﻞ اﻟﻤﺒﺎﺷﺮ ﻟﻠﺠﻠﻜﻮز واﻧﺨﻔﺎض اﻟﺴﺎﻳﺘﻮآﺎﻳﻦ IFN-γ اﻟﻤﻼرﻳﺎ اﻟﺪﻣﺎﻏﻴﻪ، اﻟﻤﻀﺎﻋﻔﺎت اﻟﻤﺘﻌﺪدﻩ، اﻟﻌﻤﺮ اﻟﺼﻐﻴﺮ وإرﺗﻔﺎع اﻟﺒﻮﺗﺎﺳﻴﻮم ﻓﻰ اﻟﺪم ﻟﺪﻳﻬﺎ إرﺗﺒﺎط ﺑﺰﻳﺎدﻩ ﻣﻀﺎﻋﻔﺎت ووﻓﻴﺎت اﻟﻤﻼرﻳﺎ. إرﺗﻔﺎع اﻟﺒﻴﻠﻮروﺑﻴﻦ، إرﺗﻔﺎع ﻋﺪد آﺮﻳﺎت اﻟﺪم اﻟﺒﻴﻀﺎء، إرﺗﻔﺎع اﻟﺒﻮﻟﻴﻨﺎ، إرﺗﻔﺎع اﻟﻜﺮﻳﺎﺗﻨﻴﻦ، اﻧﺨﻔﺎض اﻟﻬﻴﻤﻮﻏﻠﻮﺑﻴﻦ واﻧﺨﻔﺎض اﻟﺠﻠﻜﻮز ارﺗﺒﻄﺖ ﻣﻌﻨﻮﻳﺎ ﺑﺎﻟﻤﻼرﻳﺎ اﻟﺪﻣﺎﻏﻴﻪ و اﻟﻤﻀﺎﻋﻔﺎت اﻟﻤﺘﻌﺪدﻩ.