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.
VII
List of figures
Figure-1 Age distribution of UM & SM 56 Figure-5 Ratio of insulin to C-peptide in UM & SM 62 Figure-6 Correlation between blood glucose and insulin 63 Figure-7 Correlation between insulin and C-peptide 64 Figure-11 AST in UM & SM 68 Figure-12 ALT in UM & SM 69 Figure-13 ALP in UM & SM 70 Figure-16 Frequency of hyponatrmia in SM 73 Figure-17 Incidence of hypokalaemia and hyperkalaemia in UM & 74 SM Figure-18 Degree of parasitemia in UM & SM 77 Figure-19 Correlation between parasitemia and haemoglobin 77 Figure-20 Correalation between parasitemia and blood glucose 78 Figure-22 TNF-α in children with cerebral malaria and those without 79 cerebral malaria Figure- TNF-α in different malaria categories Figure-24 IFN-γ in children with severe anaemia and those without 81 severe anaemia Figure-25 Correlation of IFN-γ with haemoglobin 82 Figure-26 Correlation of IFN-γ with blood glucose 83 Figure- IFN-γ in different malaria categories Figure-28 Correlation of IL-10 with haemoglobin 85 Figure- IL-10 in different malaria categories Figure-29 TNF-α in children below and above 5 years Figure-30 IFN-γ in children below and above 5 years
VIII
List of tables Table-1 Sample characteristics 56 Table-2 The clinical presentation of children with malaria 57 Table-3 Laboratory investigations of UM& SM 58 Table-4 Comparison of RBG between UM& SM categories 59 Table-5 Comparison of insulin between UM& SM categories 60 Table-6 Comparison of C-peptide between UM& SM categories 61 Table-7 Comparison of insulin: C-peptide ratio between UM& SM 62 categories Table-8 expected value of insulin to C-peptide ratio in UM& SM 64 Table-9 Comparison of Hb between UM& SM categories 65 Table-10 Comparison of TWBCs between UM& SM categories 66 Table-11 Comparison of bilirubin between UM& SM categories 67 Table-12 Mean AST, ALT &ALP in UM & SM 68 Table-13 Comparison of AST between UM& SM categories 68 Table-14 Comparison of ALT between UM& SM categories 69 Table-15 Comparison of ALP between UM& SM categories 70 Table-16 Comparison of urea between UM& SM categories 71 Table-17 Comparison of creatinine between UM& SM categories 72 Table-18 Comparison of sodium between UM& SM categories 73 Table-19 Comparison of potassium between UM& SM categories 75 Table-20 Clinical categories of malaria and outcome 75 Table-21 The RBG, Hb, bilirubin, TWBCs and urea of 76 uncomplicated malaria and mixed presentation Table-22 The Hb, bilirubin and TWBCs of UM and cerebral malaria 76 Table-23 Comparison of period of illness between UM& SM categories Table-24 Comparison of TNF-α between UM& SM categories 80 Table-25 Correlation of TNF-α with clinical and laboratory parameters Table-26 Comparison of IFN-γ between UM& SM categories 83 Table-27Correlation of IFN-γ with clinical and laboratory parameters Table-28 Comparison of IL-10 between UM& SM categories 85 Table-29 Correlation of IL-10 with clinical and laboratory Table-30 Cytokines in children below and above 5 years Table-31 TNF-α in different malaria categories
IX CHAPTER ONE INTRODUCTION
Malaria is one of most common parasitic disease. It is caused by obligate intracellular parasites, which live in host erythrocytes and remodel these cells to provide their own needs. It is a major public health problem in tropical areas, and it is estimated that malaria is responsible for 1 to 3 million deaths and 400–900 million infections annually (1). Eighty percent of the deaths occur during the first 24 hours following admission (2, 3). The vast majority of morbidity and mortality from malaria is caused by infection with Plasmodium falciparum, although P. vivax, P. ovale, and P. malariae are also responsible for human infection. Since its discovery the problems of controlling the disease are far from being solved and endemicity is increasing. Emergence of parasite resistance to available drugs, recrudescence, recurrence and the complex pathophysiology of the infection process complicate treatment regimens.
Children of all ages living in nonmalarious areas are equally susceptible to malaria. In endemic areas, children younger than 5 years have repeated and often serious attacks of malaria. The survivors develop partial immunity at the time of adolescence (4). Older children and adults often have asymptomatic parasitemia ie presence of plasmodia in the bloodstream without clinical manifestations of malaria while the episodes of malaria and its severity are decreased. A lack of acquired immunity to P. falciparum malaria in young children appears to underlie the high rates of morbidity and mortality from malaria in areas of sub-Saharan Africa where malaria is endemic.
1 Malaria endemicity Malaria endemicity varies between different parts of the world and even within a country. It is defined by spleen rate in children aged 2-9 years. In hypoendemic areas, the spleen rate of children range is 0-10%, in mesoendemic areas the spleen rate is 10-50%, in hyperendemic areas spleen rate is 50-70% and in holloendemic areas the spleen rate is more than 70%. Malaria endemicity in Sudan Malaria is the main endemic parasitic disease in Sudan. Malaria incidence in Sudan was estimated to be about 9 million per year and the number of deaths due to malaria about 44,000. Children under 5 years of age had the highest burden (5).
Malaria transmission in eastern, western and northern states of Sudan is low to moderate, highly seasonal and occasionally epidemic. In the southern state transmission is generally perennial with moderate to high intensity. Plasmodium falciparum is the dominant species account for 95% of cases and the major anopheles vector is anopheles arabiensis (6).
Despite efforts directed toward malaria control, malaria still is a major killer of children in Africa. The pathophysiological mechanisms of severe malaria are still not fully understood. Better understanding of these mechanisms may lead to discovery of new drugs and methods of preventions. This may have great advantage in the face of increase parasite resistance to available drugs. Detecting bad prognostic indicators in severe malaria could aid clinicians caring for children with severe malaria to avoid diagnostic delays, identify the children most likely to die and thus improve management by targeting resources to children at higher risk.
2 This study will attempt to monitor physiological and metabolic variables in children with severe malaria so as to identify the more reliable indicators of serious outcomes. The ultimate aim is to consider severe malaria as an indication for intensive care. For this to take place, cost effective variables associated with fatal outcomes have to be identified. This work will look at biochemical, hormonal, haematologic and inflammatory parameters associated with morbidity and mortality.
3 LITERATURE REVIEW Malaria parasite life cycle Human malaria is caused five Plasmodium species: P. falciparum, P. vivax, P. ovale, plasmodium knowlesi and P. malariae. The life cycle of malaria parasites is complex, with asexual reproduction occurring in the mammalian host and sexual reproduction in the anopheline mosquito vector. The parasites are transmitted to humans in the form of sporozoites through the bite of a female anopheline mosquito. The sporozoites circulate for up to 45 min before entering hepatocytes, in which they undergo asexual reproduction to form a large intracellular schizont. The swollen hepatocyte eventually bursts, discharging merozoites into the bloodstream, where they rapidly invade erythrocytes to initiate the erythrocytic cycle. In P. vivax and P. ovale infections, dormant liver forms also occur that are capable of reactivating and forming a hepatic schizont weeks or months later. The blood stage merozoite is surrounded by complex membranes and contains nucleus, mitochondria, endoplasmic reticulum, ribosomes, cytosome and apical membrane organelles called rhoptries and micronemas. Inside the erythrocyte, the merozoites lose the complex membranes and apical organelles and develops within a membrane-bound parasitophorous vacuole first as a trophozoite and then, during multiple nuclear division known as schizogony, as a schizont. When the schizont matures, the host red blood cell ruptures and liberates merozoites that rapidly invade fresh erythrocytes in the general circulation, thus continuing the life cycle. Some merozoites develop into sexual forms (gametocytes), which are taken into the mosquito gut with a blood meal. These may fuse forming a zygote and then undergo meiosis to form first an ookinete and later an oocyst in the gut wall. This phase of multiplication in the mosquito is known as sporogony. The oocyst bursts,
4 liberating large numbers of sporozoites that migrate to the salivary glands to await injection into the human host during the next blood meal. Clinical presentation of malaria The spectrum of the clinical presentation and severity of P. falciparum infection is broad and vary from asymptomatic infection to severe malaria. In endemic areas, many infections in semi-immune and immune children and adults present as uncomplicated febrile illness. Nonimmune individuals may exhibit a number of complications including anaemia, coma, respiratory distress, and hypoglycemia. Many factors were found to affect clinical presentation of malaria. Transmission rate and age independently affect the clinical presentation. In areas where malaria transmission rate is high, severe malaria tends to affect children younger than 5 years while older children and adult tend to have the uncomplicated malaria. In low endemic areas, severe malaria affects older children as well (7).
Age is found to be another factor affecting the clinical presentation of malaria in children. The age distribution of the severe malaria categories is striking, but poorly understood. Children born in endemic areas are largely protected from severe malaria in the first 6 months of life by the passive transfer of maternal immunoglobulins and by fetal haemoglobin (8). The presentation of disease changes from severe anaemia in children aged less than five years to cerebral malaria in older children in areas of higher transmission (9).
The differences caused by rate of transmission and age on clinical presentation of malaria can be explained by the time of acquisition of immunity. Immunity against malaria develops slowly with increasing age.
5 Uncomplicated malaria Fever is the main presenting feature of malaria together with chills and shivering. Headache, Malaise, nausea, vomiting and diarrhea may also present. Fever is irregular at beginning of infection but if left untreated, malaria paroxysm appear. Malaria paroxysm is composed of a cold stage into which the patient feels cold due to vasoconstriction and shivering. Second stage is hot stage in which temperature rises and the skin is hot and dry. In the third stage sweating occurs and temperature falls. The paroxysm regularizes to two day cycle in plasmodium ovale and vivax (tertian malaria) while in plasmodium malariae and falciparum it regularizes to three day cycle (quartan malaria). Hepatosplenomegaly may develop during the course of the disease. Severe malaria Children with severe malaria may have the symptoms and signs of uncomplicated malaria mentioned above with one or more of WHO criteria of severe malaria (10). WHO criteria of severe malaria in children: • Cerebral malaria: unarousable coma for more than 30 minutes. • Severe anaemia: Hb less than 5 g/dl or PCV less than 15%. • Renal failure: creatinine more than 3mg/dl. • Pulmonary edema • Hypoglycemia: whole blood glucose less than 40mg/dl. • Circulatory collapse or shock: systolic blood pressure less than 50mmhg. • Repeated generalized convulsions: more than two attacks per 24 hours despite cooling. • Disseminated intravascular coagulation with laboratory evidence. • Acidemia: arterial pH less than 7.25 or HCO3 less than 15 mmol /l.
6 • Macroscopic haemoglobinuria Other features, mentioned by WHO, but do not constitute features of severe malaria are:- • Impaired consciousness but less marked than unarousable coma • Prostration • Hyperparasitemia more than 5%. • Jaundice: total bilirubin more than 3mg/dl. • Hyperpyrexia: rectal temperature more than 40°C Pathophysiology of severe malaria: The pathogenesis of severe malaria may be attributed to distinct pattern of dysregulation of the immune system to infection which can result in different disease categories of severe malaria (11). The following is a review of the role of cytokines in the pathogenesis and the immune response Overview of immunity and cytokines in malaria The immune system is a network of cells, proteins, tissues, and organs that work together to protect the body against infection or other insults. The immune system is composed of two major components: innate immunity which provides a general response against invasion by a wide range of antigens, and adaptive immunity which develops upon exposure to antigens. Cells associated with the immune system are leukocytes. There are two main categories of leukocytes; the phagocytes (granulocytes, monocytes, and macrophages) and lymphocytes. Lymphocytes are further categorized as T lymphocytes (including helper, cytotoxic, and suppressor T cells), B lymphocytes, and natural killer cells. T lymphocytes kill foreign cells through chemical lyses and help B lymphocytes in specific immune functions. B cells secrete antibodies that
7 help in foreign cells destruction. Natural killer cells target viruses and tumors. Cytokines are messengers that allow the various immune cells to communicate with each other and elicit certain responses. Cytokines are small proteins range from 5-20 KD produced by leukocytes and other cells. They differ from the classical hormones in that they are secreted by many cell types rather than specialized gland and their action is paracrine and autocrine rather than endocrine. Cytokines include interleukins, lymphokines, chemokines, some growth factors like TGF-alpha and TGF- beta and related signaling molecules such as TNF and IFN.
Immune systems use cytokines in homeostatic mechanisms and to provide signals important in host responses to invading organisms. Cytokines initiate and amplify inflammation by recruiting and activating cells, regulate the activation and differentiation of T- and B-lymphocytes and initiate and regulate local repair processes important for resolution of inflammatory responses. Cytokines act through cell receptors, which relay a signal into the cell triggering a response. Cytokine-receptors interactions are promiscuous, pleiotropic and redundant.
Cytokines member
The Interleukins
Interleukins are a group of cytokines produced predominantly by macrophages and lymphocytes.
8 Some interleukins and their functions:
Name Source Functions
-Induce acute phase inflammatory reactions& IL-1 macrophages fever. -Activate lymphocytes and phagocytes
Stimulates growth and differentiation of T cell IL-2 TH1-cells response.
IL-3 T cells Stimulates bone marrow stem cells
Has role in differentiation of B cells, IL-5 TH2-cells eosinophil production, and IgA production
macrophages, IL-6 Induces acute phase reaction TH2-cells
Involved in B, T, and NK cell survival, IL-7 - development, and homeostasis
IL-8 - Involved in neutrophil chemotaxis
Macrophags, IL-10 Suppress proinflammatory cytokines secretion TH2-cells
Involved in NK cell stimulation, Th1 cells IL-12 macrophages induction
IL-18 - Induces production of IFN-γ
9 IL-10 IL-10 is secreted by certain T lymphocytes subsets (Thelper 2 and T regulatory cells), monocytes and macrophages but the major source is the macrophage. Secretion is stimulated by endotoxin, TNF-α, cAMP elevating drugs, catecholamines and Stress. IL-10 is a homodimer made of two subunits with molecular mass of 37 KDa. IL-10 receptors found on the surface of cells and belong to cytokine receptor family type 2.
Human IL-10 was found to act as an antiinflammatory agent able to block the production of IL-6, TNF-a, IL-1 and other cytokines by monocytes/macrophages (12). This property, together with an inhibitory effect on the expression of MHC class II antigens on monocytes/macrophages results in inhibition of the antigen presenting capacity of these cells, and a consequent inhibition of T lymphocyte proliferation (13). In addition, IL-10 was recently shown to induce the proliferation of B lymphocytes and their differentiation into plasma cells secreting immunoglobulins at high rate (14, 15). Taken together, IL-10 inhibits cellular immunity reactions and stimulates humoral responses. IL-10 was also found to decrease platelet production (16).
Numerous in vitro and animal experiments suggest a major impact of IL- 10 in inflammatory, malignant, and autoimmune diseases. IL-10 over production was found in certain inflammatory disorders like rheumatoid arthritis and systemic lupus erythromatosis (17, 18). In contrast, a relative IL-10 deficiency has been observed and is regarded to be of pathophysiological relevance in certain inflammatory disorders characterized by a type 1 cytokine pattern such as psoriasis (19). Recombinant human IL-10 has been produced and is currently being tested in clinical trials. This includes psoriasis and organ transplantation
10 (19, 20). IL-10 administered to healthy volunteers was well tolerated without serious side effects at doses up to 25µg/kg, mild to moderate flu- like symptoms were observed in a fraction of recipients at doses up to 100µg/kg. A single subcutaneous dose of IL-10 resulst in transient neutrophilia, lymphocytopenia, and monocytosis and delayed decrease in platelet counts (21).
Chemokines Chemokines are small proteins secreted by cells. These molecules classified as chemokines according to structural characteristics e.g the small size of 8- 10 kilodalton and having four cystiene residues which determine the three-dimensional shape of the molecule.
They work through paracrine chemotaxis and interaction with G protein- linked transmembrane receptor on target cells. Chemokine receptors are G protein coupled receptors containing 7 transmembrane domains that are found on the surface of WBCs. Activation of G proteins causes the subsequent activation of an enzyme phospholipase C(PLC). PLC cleaves Phosphatidylinositol (4,5)-bisphosphate (PIP2) into Inositol triphosphate (IP3) and diacylglycerol (DAG) that trigger intracellular signaling events; DAG activates another protein kinase C (PKC), and IP3 triggers the release of calcium from intracellular stores. This in turn leads to activation of many cellular pathways which generate responses like chemotaxis, degranulation, release of superoxide anions and changes in the avidity of cell adhesion molecules called integrins within the cell having the chemokine receptor.
11 Interferons
Interferons (IFNs) are proteins produced by virally infected host cells in response to viral double stranded RNA. Different types of IFNs are produced by different types of cells; machrophages are the primary producers of IFN-α and IFN-β, whereas T lymphocytes release IFN-γ
There are three types of interferons classified according to the type of receptor through which they signal; Interferon type I which binds to the IFN-α receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains, Interferon type II binds to IFNGR and interferon type III which signal through a receptor complex consisting of IL10R2 and IFNLR1
The JAK-STAT signaling pathway is the best-characterized IFN signaling pathway. interferons are antiviral and possess antioncogenic properties, macrophage and natural killer lymphocyte activation, and enhancement of major histocompatibility complex glycoprotein classes I and II, and thus presentation of foreign peptides to T cells. The production of interferons is induced by microbes as well as mitogens and other cytokines.
Tumor necrosis factor-alpha
TNF-α is mainly produced by macrophages, but also by a wide variety of cells including lymphoid cells, mast cells, endothelial cells, cardiac myocytes, fibroblasts and neuronal tissue. Large amounts of TNF-α are released in response to lipopolysaccharide, other bacterial products and IL-1.
12 TNF-α is primarily produced as a 212 amino acid-long transmembrane protein arranged in stable homotrimers. From this membrane-integrated form the soluble homotrimeric cytokine (sTNF) is released via proteolytic cleavage by the metalloprotease TNF alpha converting enzyme.
Two receptors, TNF-R1 (TNF receptor type 1) and TNF-R2 (TNF receptor type 2), bind to TNF. TNF-R1 is expressed in most tissues, and can be activated by both the membrane-bound and soluble forms of TNF- α, while TNF-R2 is only found in cells of the immune system and respond to the membrane-bound form of the TNF. Binding of TNF with their receptor, leads to a conformational change to occur in the receptor, leading to changes in transcription of many proteins involved in cell differentiation, proliferation and inflammation.
TNF-α is involved in systemic inflammation and is a member of a group of cytokines that all stimulate the acute phase reaction. TNF-α causes apoptotic cell death, cellular proliferation, differentiation, inflammation and ihibits tumorigenesis and viral replication. It has anti-apoptotic activity as well.
It has a number of actions on various organ systems, generally together with IL-1 and Interleukin-6: On the hypothalamus TNF-α stimulates the hypothalamic-pituitary-adrenal axis by stimulating the release of corticotropin releasing hormone (CRH) and induction of fever (22, 23).
On the liver, TNF-α stimulates the acute phase response, leading to an increase in C-reactive protein and a number of other mediators (24). It also induces insulin resistance by promoting serine-phosphorylation of insulin receptor substrate-1 (IRS-1), which impairs insulin signaling (26). On other tissues TNF-α increases insulin resistance (25, 26).
13 In the immune system, TNF-α was found to attract neutrophils very potently, and helps them to stick to the endothelial cells for migration as well as enhancing their phagocytostic capacity (27, 28).
Dysregulation and overproduction of TNF-α have been implicated in the pathogenesis of many human diseases (29, 30)
General properties of cytokines receptors
Cytokines act on their target cells by binding specific membrane receptors. Cytokines interactions with their receptors are characterized by being non-specific, pleiotropic, redundant and Polyspeirism. Non- specificity (promiscuity) means that the binding site of a receptor can bind many ligands and a ligand can bind several receptors. The pleiotropic characteristic indicates that a single cytokine molecule can produce many diverse effects. IFN-γ activates >200 different genes. Redundancy means that several agents can perform the same action. Although the agents are not identical, they are redundant to the degree to which they may replace one another functionally. Chemokines are example of a redundant cytokine system. Polyspeirism is a state in which multiple cytokines are produced in a redundant way by a single cell together in response to the same stimulus.
Effects of cytokines on the immune system Cytokines mediate immunity in three ways; first they mediate natural immunity, included in this group IL-1, TNF-α, IFN-α and IL-8. Some of these cytokines eg IFN-γ protect against viral infection whereas others eg IL-1, TNF- α initiate non specific inflammatory response. Secondly, cytokines stimulate lymphocytes growth, differentiation and activation. Within this group, IL-2, IL-4, IL-5, IL-12 and TGF-β. Thirdly, cytokines
14 activate inflammatory cells .Within this group, IFN-γ, TNF-α, TNF-β, migration inhibitory factor and IL-8.
To responses in a timely and efficient manner, the immune system developed numerous mechanisms, including T regulatory cells (Tr cells), to modulate and down-regulate immune responses at different stages. Tr cells include regulatory Th2 cells (which suppress Th1 associated responses), Th1 cells (which suppress Th2-cell-mediated responses), IL- 10-producing Tr1 cells (a subset of IL-10-dependent, antigen-specific regulatory T cells), TGF-β1-secreting Th3 cells, CD8+, natural killer T, and γδ T cells. Several cytokines including IL-10 and TGF-β1 have been implicated in Tr effector functions.
IL-10 down regulates T helper 1 responses, namely IFN-γ secretion and activation of monocytes and macrophages. IL-10 controls also Th2- mediated inflammatory processes. IL-10 also has immunostimulatory properties especially on B cells and activated CD8+T cells. However, IL- 10 is able to induce the differentiation of a subset of regulatory CD4+T cells (Tr1) and is important for the in vivo function of regulatory T cells and these supports the role of IL-10 as an important cytokine in the control of immune responses. In different experiments, these cells were shown to inhibit Th1 and Th2-type inflammatory responses through the secretion of IL-10. These Tr1 cells may thus have important pharmacological uses in inflammation. Cytokines and immunity in malaria Following repeated exposure to infection, people living in malaria endemic areas gradually acquire mechanisms to limit the inflammatory response to the parasite that causes the acute febrile symptoms as well as mechanisms to kill parasites. Children, who have yet to develop
15 protective immune mechanisms are thus at greater risk of clinical malaria, severe disease and death than adults. Artaavanis-Tsakonas et al (2003) suggested the following model of antimalarial immunity; in endemic populations, primary malaria infections in infants induce low levels of IFN-γ and TNF-α via an innate pathway and at the same time antigen specific T cells are primed. The infection induces minimal clinical symptoms and parasites are cleared, either immunologically (via opsonization by maternal antibody or cytokine-mediated parasite killing) or because parasites fail to establish themselves in the face of physiological barriers (e.g. fetal haemoglobin and dietary deficiencies) (9). On re-infection, the malaria primed T cells produce greatly increased amounts of IFN-γ which synergizes with malarial glycosylphosphatidylinositol to up-regulate the production of TNF-α leading to an increased risk of severe disease. Further infections induce effective antiparasitic immunity which reduces the parasite load and thus the concomitant level of antigenic stimulation, thereby dampening the pro-inflammatory cytokine cascade. Falling antigen concentrations or other factors lead to a switch in the predominant T cell phenotype from Th1 (IFN-γ producing) to a regulatory T cell phenotype (IL-10 and TGF- β producing). Thus immune individual can clear the infection without running the risk of overproducing dangerous inflammatory mediators. The risk of severe disease may thus depend on the relative speed with which the different components of the antimalarial immune response develop (31). However cerebral malaria, a common manifestation of severe malaria, occurs in children who have probably acquired a significant degree of antimalarial immunity as evidenced by higher levels of circulating antimalarial antibodies (32). The potential explanation is that cerebral malaria is, in part, an immune-mediated disease in which αβ T cells
16 priming occurs during first infection and eventually leads to excessive IFN-γ and TNF-α production and immunopathology on re-infection (33)
Both innate and adaptive immunity are involved in immunity against malaria. Innate immunity: Innate immunity is thought to play an important role in clearing Plasmodium from the infected hosts. Most of the elimination occurs in the spleen, although the liver has been shown to be an alternative site (34). Cells from the monocyte and macrophage are more effective than neutrophils in phagocytosing parasitized erythrocytes (35). Macrophages of the red pulp carry out this job specifically (36). Macrophage is activated by natural killer cells (NK) and γδ T cells-derived IFN-γ enhancing phagocytosis and killing of parasitized erythrocytes (37). Recent studies have shown that P. chabaudi infection in γδ T-cell deficient mice have exacerbated early and chronic parasitemias (38). These results are in accordance with other studies that describe early production of IFN-γ and TNF-α both to splenic γδ T lymphocytes and to NK cells (39). These results suggest that the early production of both cytokines is important in the early control of parasitemia and that both natural killer and γδ T cells contribute equally towards their production. IFN-γ produced by NK cells are detectable within 6 h of co culture with infected RBC, their numbers peaking at 24 h in most donors. There is marked differences between donors in magnitude of the NK-IFN-γ response. This difference between donors suggests a variation in their ability to mount a rapid proinflammatory cytokine response to malaria infection that may, in turn, influence their innate susceptibility to malaria infection and its complications (40). Peripheral blood mononuclear cells taken from African children presenting with uncomplicated malaria
17 disease were more efficient producers of IFN-γ when stimulated with sporozoite or merozoite antigen peptides in contrast to cells taken from children with severe malaria (41). Furthermore, hyperparasitemia was associated with a lower frequency of producing CD4+Tcells in Gabonese patients presenting with acute P. Falciparum malaria (42).
In murine model, enhanced NK cell activity were detected in spleens of mice infected with irradiated Plasmodium berghei sporozoites was detected (43). More recently, Plasmodium yoelii sporozoite infection has been shown to induce a rapid inflammatory response in the liver characterized by NK cell, macrophage, and T cell infiltration and IFN-γ production (44). It has been proposed that protective immunity to P. yoelii liver stages mediated by parasite-specific CD8+ T cells is dependent on the presence of IL-12 and NK cells (45), indicating an important synergy between innate and adaptive immunity in this system. Innate immunity may have a role in immunity against blood stages malaria as depletion of NK cells from Plasmodium chabaudi-infected mice results in a more severe course of infection with higher parasitemia and increased mortality (46).
Studies in both mice and humans have shown that proinflammatory cytokines, specifically IL-12, IFN-γ and TNF-α, are essential mediators of protective immunity to erythrocytic malaria (47, 48). Resistance to rodent malaria is found to be dependent on signals mediated by IFN-γ (47), and the difference between lethal and nonlethal infections depends on the ability of the mouse to generate an early IL-12, IFN-γ or TNF-α response (48, 49, 50, 51). TNF-α and IFN-γ act synergistically to optimize nitric oxide (NO) production (52), which in turn leads to parasite killing (53). In humans, IFN-γ production is correlated with resistance to reinfection with Plasmodium falciparum (54, 55) and
18 protection from clinical attacks of malaria(56), plasma TNF-α and nitric oxide levels are associated with resolution of fever and parasite clearance (57, 58), and plasma TNF-α and IFN-γ mediate loss of infectivity of circulating gametocytes (59). Many vaccine developers now regard IFN-γ production to be the hallmark of effector T cell function for malaria (60, 61).
The role of nitric oxide in malaria:
NO is formed by the oxidative deamination of the amino acid L-arginine by nitric oxide synthases (NOS). Three isoforms of this enzyme have been described, Neuronal NOS (nNOS or NOS1). NO is naturally found in both the central and peripheral neuronal cells, where NO is neurotransmitter. Endothelial NOS (eNOS or NOS3) is expressed by the endothelium and other cell types and is involved in cardiovascular homeostasis. In contrast, inducible NOS (iNOS or NOS2) is not found in resting cells, but its gene is rapidly expressed in response to stimuli such as proinflammatory cytokines. When induced, iNOS synthesizes 100- 1000 times more NO than the constitutive enzymes and for prolonged periods. This high concentration of NO may inhibit many microbes, but may also damage the host, thereby contributing to pathology. NO inhibits the growth of many bacteria and parasites in vitro. The antimicrobial effect is not from NO itself, but from reactive nitrogen intermediates formed by the oxidation of NO. For example, reaction between NO and the free radical superoxide (O2 ) results in the formation of the unstable molecule peroxynitrite (OONO ). These reactive nitrogen intermediates inactivate key microbial enzymes, such as ribonucleotide reductase and aconitase, by reacting with iron containing groups in these enzymes.
19 Nitric oxide has been shown to be protective against Plasmodium falciparum in vitro. NO levels were inversely related to disease severity, with levels highest in subclinical infection and lowest in fatal cerebral malaria (62). NO appears central to the host response in malaria, mediating the antiparasitic effects of proinflammatory cytokines at each stage of the parasite life cycle in both experimental and possibly human infection (63). In addition to its protective role, NO have been suggested as a mediator in cerebral malaria. NO production requires tight control to limit cytotoxic damage to the host's own cells. Unregulated production may lead to a variety of damaging effects including alterations to normal neurological functions during cerebral malaria (64).
CSF NO intermediates (nitrite, nitrate), which may be more reflective of local cerebral production and is less affected by diet and other confounders, is increased in cerebral malaria(65). Immunohistological studies suggest widespread iNOS induction in cerebral endothelium and resultant NO production. Different genetic polymorphisms of the iNOS promoter region have been associated with increased risk of death from cerebral malaria in Gambian children(66) and with protection from severe malaria in Gabonese children(67) suggesting that complex genetic factors may determine iNOS production and thereby influence clinical outcome.
Adaptive immune response: Both the cellular and humoral arms of the adaptive immune system are important elements in the eradication of Plasmodium from the body, and both are dependent on αβ CD4+ lymphocytes. Both Th1 and Th2 responses are required in controlling of the infection, but they need to be adequately tuned in intensity and time (68, 69). Both Th1 and Th2 are activated during infection with P. yoelii in mice, but early activation of
20 Th2 cells leads to increased susceptibility to infection (70). In addition, in P. chabaudi chabaudi AS infection there is a shift from Th1 to Th2 during peak parasitemia that is important for clearance of parasites (71).
Treatment with anti-TNF-α monoclonal antibody results in a tendency toward longer times for parasite clearance. Interestingly, this effect is associated with reduced levels of IFN-γ (72).
IL-18 was found to enhance Th1 immune responses through IL-12 and also by potentiation of Th2 immune responses in the absence of IL-12. In malaria, the role of IL-18 in early immunity against the parasite has recently been suggested that as protective by enhancing IFN-γ production in vivo (73).
Early IL-10 production has been associated with susceptibility to infection (69, 74), and it is thought that this cytokine has a prominent anti- inflammatory effect, limiting in some way the damage which occurred on normal tissues by an excessive Th1 response.
Studies with transforming growth factor β (TGF-β), another anti- inflammatory cytokine, resulted in contradictory findings. On the one hand, positive effects of TGF-β on immune responses against Plasmodium have been described. Anti-TGF-β worsened P. berghei infection and conferred susceptibility to P. chabaudi but not to P. yoelii (75). On the other hand, resistant mice became susceptible to P. chabaudi upon administration of recombinant TGF-β, whereas susceptible mice became resistant upon administration of anti-TGF-β, with concomitant increases in IFN-γ and NO production (76)
21 Cytokines in the immunopathology of malaria
The pathogenesis of malaria is multifactorial and most probably involves immunologic and nonimmunologic mechanisms (77). The parasite itself can contribute to the severity of disease, as it has an ability to infect a high percentage of erythrocytes (78) and to induce production of proinflammatory cytokines. Glycosylphosphatidylinositol toxin from Plasmodium is an important pathogenic factor due to its ability to induce TNF-α and IL-1 (79).
Severe malaria has been associated with high circulating levels of inflammatory cytokines such as TNF-α, IL-1 and IL-6. TNF-α is higher in plasma from children with CM or severe anaemia than in plasma from UM malaria cases (80-82). IFN-γ has been linked to the onset of pathology in mice as well as in humans. The detrimental effects of IFN-γ are believed to be due to its ability to activate macrophages which, in turn, produce endogenous pyrogens (TNF-α, IL-1and IL-6) leading to an inflammatory cascade (83, 84). IFN-γ is essential for the onset of CM as IFN-γ receptor deficient mice do not develop CM (85).
Other example of the role of cytokines in pathogenesis of severe malaria is in severe anaemia. The levels of IL-12, a cytokine that boosts erythropoiesis, are correlated with anaemia (86). Furthermore, administration of recombinant IL-12 is able to ameliorate anaemia in mice infected with P. chabaudi (87). Recently, production of macrophage inhibitory factor, another factor produced by macrophages, has been correlated with the development of anaemia in mice (88). Higher levels of TNF-α in serum were found to be associated with severe anaemia (89). However, this is not a universal finding. In another study, low levels of
22 this cytokine were detected in the serum of Ghanaian children with severe anaemia (90). This may be explained by the fact that cytokine balance is found to predict malaria clinical presentation. IL-10 was found to be low in severe anaemia and TNF-α: IL-10 ratio was higher in children with severe anaemia than in other malaria complication (82, 91). So, antiinflammatory cytokines are required to down regulate the pathological effects of high concentrations of the proinflammatory cytokines. Serious complications of malaria The following pages will present the more serious common complications of severe malaria and the present knowledge of their pathophysiology. Cerebral malaria Cerebral malaria is a leading cause of death in severe malaria. Malaria affects the brain in two ways; either direct brain involvement or indirect through high grade fever, hypoglycemia, hyponatremia and severe anemia leading to various neurological manifestations. World health organization (WHO) defined cerebral malaria as an arousable coma for more than thirty minutes in patients with positive blood film for malaria. The coma is usually preceded by fever and convulsions (92). These convulsions can be generalized tonic- clonic or focal and are associated with poor outcome (92, 93). Most children survive with appropriate management but about 10.9% recover with neurological and cognitive impairments (92, 94, 95, 96). Deaths occur within the first day of admission (92).
Gross examination of the brain of patients who died of CM revealed edematous swollen brain with petechial and subarachnoid hemorrhages. Microscopically, there is damage of the vessel walls and blood brain
23 barrier disruption, enlarged perivascular spaces and adherence of leucocytes and parasitized RBCs to brain vessels. Scanning electron microscopy of these patients revealed the same feature and the adhered leucocytes are monocytes and lymphocytes (97)
The pathogenesis of cerebral malaria is still not well known. Magnetic resonance study of experimental CM suggests inflammatory, ischemic process and edema role in fatal CM. Vascular damage and blood brain barrier disruption are the cause of hemorrhages and edema which contribute to reduction in the brain perfusion and ischemia. Reduced high energy phosphate and high brain lactate together with edema which worsens the ischemia by further compressing the arteries was reported (98). Another study revealed metabolic changes in CM consistent with brain ischemia rather than hypoxia (99). Increased vascular permeability and subtle changes in BBB integrity were reported in CM (100, 101, 102). Other mechanisms of the reduction of the blood flow involve endothelin. Endothelin is a 21 peptide synthesized by the endothelial and others cells. Its expression is augmented by stressful stimuli to the endothelium. It is a potent vasoconstrictor. In CM where endothelium is damaged, it provides a stimulus to increased expression of endothelin which in turn induces brain vessels constriction leading to ischemia and neuronal damage (103). All these finding support ischemia and the inflammatory process as the cause of CM.
Other studies attribute CM pathogenesis to sequestered lymphocytes and parasitized RBCs leading to cerebral hypoxia. An ultra structural study of the brain carried on Thialand and Vietnam on 65 patients who died of severe malaria revealed higher level of sequestration of parasitized RBCs in the brain vessels of patients with CM compared with those without CM and the degree of sequestration in the brain vessels is positively
24 correlated with premortem coma (104). Depletion of alpha and beta lymphocytes protects against CM (102). Lymphocytes induce local production of IFN-γ, TNF-α and NO which are known to have a pathological role in CM (105). Depletion of T regulatory cells in mice was found also to protect it from CM. The mice had reduced numbers of active CD4 and CD8 T cells in the spleen and lymph nodes with selective reduction of CD8 T cells in the brain suggesting T regulatory cells contribution to pathogenesis of CM by modulating the immune response (105). Platelets were found to accumulate in brain vessels of patients dying of CM and may play a role by providing adhesion receptors involved in sequestration (107).
The sequestration of parasitized RBCs and WBCs in the brain vasculature result in obstruction of the blood flow and hypoxia leading to neuronal dysfunction as apparent from degeneration of the neurons and high CSF lactate (108).
Sequestration is a mechanism of protection for the parasite to escape from phagocytic activity of the spleen and provide good media for parasite growth as sequestration occur in capillaries and venules (109). Sequestration of parasitized RBCs involves adhesion between the parasitized RBCs and the vascular endothelium (110, 111). At the time of entry of merozoite to the RBC, it inserts antigens on the RBC surface membrane. These antigens appear as irregularities or protrusions in the RBCs surface membrane and are known as knobs. There are three proteins involve in the knobs; histidine-rich protein (PFHRP-1) and plasmodium falciparum erythrocyte membrane protein-1 and -2 (PFEMP- 1, PFEMP-2) (112, 113, 114). Through these knobs the parasitized cells adhere to the endothelial receptors (platelet glycoprotein-IV, intercellular adhesion molecules-1(ICAM-1), vascular cell adhesion molecules-
25 1(VCAM-1), thrombospondin, E-selectin and chondroitin sulphate (115, 116, 117, 118). Some of these endothelial receptors are constitutively present; some are enhanced by cytokines while others are induced by cytokines. Platelet glycoprotein-1V (CD36) is found on platelet, endothelial cells, monocyte and erythroblast. ICAM-1 molecules are present in most capillaries endothelium however its expression was found to be upregulated by TNF, IFN and IL-1 during CM (119). TNF, IL-1 and IL-4 induced E-selectin and VCAM-1 expression in the vascular endothelium and are not constitutively expressed on the vascular endothelium (120). Chondroitin sulphate is a recently discovered adhesion molecules involved in the sequestration of the parasite in the placenta (118). Ninety percent of adhesion of the parasitized RBCs to the endothelium involves CD36 and ten percent to ICAM-1 while adhesion to the other adhesion molecules is minimal (121). The adhesion process occurs in three steps; tethering, rolling then firm attachment (122). Some of the adhesion molecules facilitate the process of adhesion of the parasitized RBCs to other adhesion molecules eg ICAM-1 facilitates adhesion to CD36 (122). Still the binding to CD36 can occur directly (124). Some of the endothelial receptors are constitutively expressed and show no increase in severe disease while others are induced during the course of the disease and showed to be high in patients with CM (125).
Increased RBCs rosetting and decreased deformability of the RBCs were reported as contributing factors in obstruction of the cerebral blood vessels. Rosetting is a mechanism by which parasitized RBCs adhere to non-parasitized RBCs. Increased rosette formation was detected in patients with CM (126) and the sera of those patients are deficient of anti- rosetting antibody (127). Decreased deformability of parasitized RBCs
26 was found to be a strong indicator of unfavorable prognoses in severe malaria (128).
The other supposed mechanism of CM pathogenesis involves cytokines. Early production of cytokines was found to be protective against CM but late production is associated with increased risk of getting CM. In mice, infection with plasmodium berghei ANKA results in production of IFN-γ at 4 day postinoculation and CM on day 6, while infection with P. berghei K173 is associated with production of IFN-γ, IL-10 and IL-12 24 h postinoculation but does not result in CM. When mice were co-infected with both ANKA and K173, they produced an early cytokine response, including a burst of IFN-γ at 24 h post inoculation. These co- infected mice failed to develop CM (129).
TNF-α is a key cytokine in the pathogenesis of CM as high serum concentration of TNF-α and its two soluble receptors STNF R1, R2 were detected in patients with CM (130). Augmented expression of TNF-α and IFN-γ were also found in the brains of patients with CM (131, 132). Consistent with these findings, experimental and clinical studies revealed that serum TNF-α level is high at the onset of neurological signs and that passive immunity against TNF-α significantly increased mouse survival rate and prevented the histopathological changes which characterize CM and the appearance of the neurological signs (133). Clinical data obtained from African children with CM showed elevated levels of TNF-α are associated with severe neurological involvement and fatal outcomes (81).
TNF-α was thought to cause CM through many mechanisms. TNF-α upregulales the expression of the adhesion molecules involved in sequestration (119, 133). It is responsible for many of the histological
27 features associated with CM in mice like degeneratrion, oedema and haemorrhages (133)
TNF-α was also found to induce NO production in the brain during CM (134). NO is known to affect synaptic transmission and thus it may contributes to the seizure and coma (135). Increased expression of inducible NO synthase was detected in the neurons, microglial cells, astrocytes and in the macrophages surrounding ring hemorrhages. Its expression is decreased in the recover CM and there is positive correlation between the intensity and the number of iNOS positive vessels and severity of malaria related histopathological changes (135). NO metabolites were found to be higher in plasma and the CSF of children with CM and associated with poor outcome (137, 65, 138).
Microglial cells activated by TNF-α were found to undergo morphological changes during CM like soma enlargement, retraction of the ramified process, increased amoeboid appearance and vacuolation which are consistent with microglial cells activation. The activated microglial cells redistribute toward vessels (139, 140). Microglial cells are thought to cause CM pathology through excitotoxic mechanism as increased quinolonic acid concentration was found to be high in the CSF of patients with CM and associated with increased severity of the disease (141).
IFN-γ contributed to CM pathogenesis through augmentation of both the production and the action of TNF-α. Monoclonal antibodies against IFN- γ prevent the occurrence of CM as well as preventing elevation of TNF-α (142). In addition to its ability to up-regulate TNF-α production, IFN-γ also promotes the quinolinic acid pathway of tryptophan metabolism by inducing the enzyme indoleamine 2, 3 dioxygenase. Quinolinic acid is a
28 neurotoxic may also contribute to the development of CM. Indoleamine 2,3 dioxygenase activity has been reported to be 5 fold higher in mice with cerebral malaria than in those without cerebral symptoms and associated with a marked increase in the Quinolinic: kyurenic acid ratio (143). Raised Quinolinic: kyurenic acid ratio was reported in Vietnamese adults with cerebral malaria with especially high ratio in those who died (144).
On the other hand, IL-10 is protective against CM development by inhibiting TNF-α as IL-10 knockout mice infected with plasmodium chabaudi develop greater parasitic sequestration and severe cerebral edema and hemorrhages and administration of anti-TNF-α ameliorate both cerebral edema and haemorrhages (145).
In contraversery, anti TNF-α antibody did not affect the outcome of children with CM in some studies (146). NO metabolites were low in both the plasma and the CSF of children with CM (141, 147). NO is thought to has a protective role in CM by inhibiting brain indoleamine 2/3 dioxygenase induction and quinolinic acid accumulation during hypoxia (148).
Repeated convulsions
WHO defines repeated convulsions as convulsions more than 2 within 24 hours despite cooling. It is the most prevalent complication in young children (149). Repeated convulsions were found to indicate cerebral dysfunction rather than being febrile convulsions as no correlation between body temperature and convulsions was detected. It may be the early warning signs of cerebral involvement (150).
29 An increased prevalence of epilepsy was reported in children previously admitted with CM and repeated convulsions compared with the unexposed group. The most commonly encountered seizure types were tonic–clonic, focal becoming secondarily generalized and both. Twenty- six percent of children who developed epilepsy had EEG abnormalities at time of severe malaria. The prevalence of epilepsy associated with malarial repeated convulsions is more than twice that reported after complicated febrile seizures (151). Severe anaemia Severe anemia is one of the major manifestations of severe malaria. WHO defines severe anemia as hemoglobin level below 5g/dl. It is usually normocytic normoochromic anaemia. In children, it is commonly encountered below 5 years of age (9).
Severe anaemia was found to contribute by 53% of malaria related deaths in study carried in Kenya (151) while some studies revealed that severe anaemia mortality rate increased only when it is combined with other malaria complications like cerebral malaria and respiratory distress; but it is not a predictor of fatal outcome if it is the only presentation (153).
The pathophysiology of severe anaemia is complex and multifactorial. Malarial anaemia is thought to arise from both decreased red blood cell (RBC) production and increased RBC destruction (31). Destruction of RBCs can occur as a result of parasite invasion and replications. The parasite lives in red blood cells, use haemoglobin for their growth and replication and at the end schizonts rupture and destroy their erythrocyte host cells, although some parasites may be removed from erythrocytes as immature ring forms by phagocytic cells (154). Anaemia correlates with the degree of parasitemia and duration of the acute illness (155, 156). The
30 deformability of the RBCs is reduced in severe malaria and provides another signal for clearance of RBCs by spleen (157).
Infected erythrocytes may also be phagocytosed by macrophages following opsonization by immunoglobulins and/or complement components (158, 159, 160). Circulating immune complexes were detected in malaria which leads to activation of the complement system inducing removal of the RBCs through phagocytosis in the liver and the spleen (158). Complement system activation resulting from binding of IgG to plasmodium falciparum erythrocyte membrane protein-1(PFEMP- 1) is reported (158, 161, 162). Furthermore children with severe malarial anemia were found to have reduced levels of complement regulatory proteins (complement receptor1, decay accelerating factor and membrane inhibitor of reactive lysis) leading to increased susceptibility of RBCs to complement deposition (159).
Destruction of the RBCs is not confined to the infected ones but uninfected RBCs are also destroyed by the immune system and it may be the major cause of haemoglobin loss (163). Immunoglobulin was found to bind uninfected RBCs enhancing their phagocytosis(164). Ring surface protein (RSP) is a parasite derived protein inserted on the surface of the RBCs in which the parasite fails to invade. Anti- RSP antibodies were detected in the serum of patients with severe anaemia and were found to enhance phagocytosis and destruction of this RSP- bound RBCs (165). Furthermore, this antigen was detected in RBCs precursors in the bone marrow leading to its destruction by complement mediated phagocytosis (165). Uninfected RBCs were also found deficient in complement regulatory protein leading to activation of complement system (160).
31 Sometimes in asymptomatic malaria, anaemia is frequently out of proportion to the low level of parasitaemia found (167), suggesting that it is not mediated simply by direct haemolysis of parasitized red blood cells. Reticulocytopenia has been observed in numerous clinical studies of malarial anaemia (168, 169). Suppression of erythropoeisis and dyserythropoeisis are reported in malaria (168). The histopathological study of the bone marrow of children with malarial anaemia shows impaired bone marrow response ( 170, 171, 172), erythroid hyperplasia, with dyserythropoiesis, cytoplasmic and nuclear bridging and irregular nuclear outline(168).
Parasite factors, nonoptimal response to erythropoietin and proinflammatory cytokines may affect bone marrow function and contribute to the development of severe anaemia in malaria. Hemozoin, a product of heme digestion by the parasite, is supposed to inhibit erythropoeisis in malaria. In children with malarial anaemia, the proportion of circulating monocytes containing hemozoin is found to be associated with anaemia, and this is independent of the level of circulating cytokines, including TNF-α (173). Histological examination of the bone marrow of children who died from malaria showed that pigmented erythroid and myeloid precursors are associated with the degree of abnormal erythroid development (173). Another way by which hemozoin inhibits erythropoeisis is through induction of NO production (174). NO was found to suppress erythropoiesis (175) and to induce apoptosis in erythropoietic precursors (176).
Erythropoietin, a hormone produced by the kidney and the liver, stimulates RBCs production. In malaria, erythropoietin level is appropriate to degree of anaemia but the response of the bone marrow is
32 limited as apparent from inadequate reticulocytosis. There is suboptimal proliferation of erythroid progenitor cells leading to suboptimal number of erythroblast. Also the maturation and differentiation of this erythroblast is suppressed (177, 178).
There are controversial findings regarding the role of TNF-α, IL-10 and IFN-γ in pathogenesis of severe anaemia in children. TNF-α inhibits all stages of erythropoiesis (179) and IFN-γ works with TNF-α to inhibit erythroid growth and differentiation by up-regulating expression of TRAIL, TWEAK, and CD95L in developing erythroblasts (180). In children with severe anaemia, TNF-α was found to be high while IL-10 was low (181, 182, 183). However, in a nonlethal P chabaudi infection of mice, neutralization of TNF-α or IFN-γ has no effect on in vitro erythropoiesis (184). The severity of anaemia seems to be dependent on levels of TNF-α relative to its regulator, the potent anti-inflammatory cytokine IL-10. Several clinical studies have demonstrated that a low ratio of plasma IL-10/TNF-α is associated with severe malaria anaemia in young children (82, 91). IL-10 knock-out mice infected with P chabaudi display increased anaemia (184) which was reversed following TNF-α neutralization in vivo (186). Thus IL-10 may protect against the bone marrow suppression and the erythrophagocytic activity induced by TNF-α.
Some studied, however, reported that IFN-γ is potential marker of protective immunity against severe anaemia in malaria. A study conducted in Kenyan children aimed to assess IFN-γ responses to six preerythrocytic antigens detected that IFN-γ positive responders had higher haemoglobin levels and significantly reduced prevalence of severe anaemia compared with IFN-γ nonresponders, suggesting that IFN-γ
33 immune responses to these preerythrocytic antigens were associated with protection against malarial anaemia (187).
Anaemia in falciparum malaria is clearly multifactorial and there is a strong argument that erythrocyte destruction and ineffective erythropoiesis play equal parts in the etiology of malarial anaemia.
Hypoglycemia WHO defines hypoglycemia as blood glucose level below 40mg/dl. Hypoglycemia is found to be associated with increased morbidity and mortality in malaria (188, 189). It is common in children less than five years of age (190, 191).
The pathophysiology of hypoglycemia in malaria is not well understood probably it is a multifactorial. Glucose consumption by the parasites, hyperinsulinemia, gluconeogensis insufficiency and cytokines were all supposed to play a role.
The malaria parasite exhibits a rapid growth and multiplication rate during many stages of its life cycle. This necessitates that the parasite acquires nutrients and metabolize these various biological molecules in order to survive and reproduce. The parasite depends on glucose as a primary source of energy and it take it from host blood stream (192). Woodrow et al (1999) discovered a parasite-encoded hexose transporter that is localized to the region of the parasite plasma membrane within the infected red cell. It is a facilitative transporter with relatively high affinity for glucose. This transporter is presumed to act in conjunction with important changes in the permeability properties of the infected erythrocyte membrane to regulate the uptake of substrates by parasites (193). The parasite exhibits a high rate of glycolysis and utilizes up to 75
34 times more glucose than uninfected erythrocytes. Most of the glucose is converted to lactate and the high lactate dehydrogenase (LDH) activity is believed to function in the regeneration of NAD+ from NADH which is produced earlier in the glycolytic pathway by glyceraldehyde-3-phophate dehydrogenase and about 85% of glucose is converted to lactate which is exported from the infected cells (194).
Some studies detected hyperinsulinemia at times of hypoglycemia and reporting that hyperinsulinemia may be the cause of low blood glucose levels (195, 196). Elased et al (1994) reported that administration of diazoxide, an insulin secretion inhibitor, prevents development of hypoglycemia (195). Parasite derived factors and quinine treatment were found to stimulate insulin secretion (195, 196, 197, 198, 199). However, another study reported appropriated insulin response in children with hypoglycemia (189, 191)
Gluconeogenesis insufficiency is also reported to play a role in hypoglycemia pathogenesis. English et al (2008) investigated the potential importance of glycerol as a substrate for gluconeogenesis and whether substrate limitation contributes to hypoglycaemia in severe malaria in African children. About 16% were hypoglycaemic on admission, while at least 9% of children with severe malaria treated with quinine and a concurrent 4% dextrose infusion had a definite episode of hypoglycaemia after admission. Blood levels of gluconeogenic precursors (alanine, glycerol and lactate) levels were high in those with either hypoglycaemia on or after admission as they are in children not having an episode of hypoglycaemia. However children with severe malaria with hypoglycaemia at some stage are more acidotic and suffered from renal
35 impairment than those who are not hypoglycaemic. The study concluded that gluconeogenesis is impaired in severe malaria (197).
Taylor et al (1989) assessed the importance of both pretreatment and quinine-related hypoglycemia in children in an area in which the disease is endemic in 95 Malawian children with falciparum malaria and altered consciousness who were treated with intravenous quinine. Hypoglycemia was associated with low plasma insulin concentrations and with elevated plasma concentrations of lactate, alanine, and 5'-nucleotidase which suggests impaired hepatic gluconeogenesis (189).
In contrary to the English and Taylor studies, a study carried by Dekker et al aimed to measure basal glucose production and gluconeogenesis and estimate the flux of the gluconeogenic precursors in Kenyan children with uncomplicated falciparum malaria before and during infusion of alanine. Alanine infusion increased basal plasma glucose concentration suggesting that glucose production in children with uncomplicated falciparum malaria is potentially limited by insufficient precursor supply leading to failure to compensate in the presence of decreased glycogen flux to glucose, increasing the risk of hypoglycemia (191).
The role of cytokines in pathogenesis of hypoglycemia is controversial. TNF-α is believed to increase rather than decrease blood glucose level (195). TNF-α was found to inhibit insulin secretion and increased peripheral resistance to insulin (24, 25, 195) leading to increased blood glucose level. On the other hand some studies detected high TNF-α, IL- 10 and IL-6 levels to be associated with hypoglycemia (191, 196, 200).
Metz et al (1999) conducted a study in six healthy subjects to evaluate the endocrine and metabolic effect of IFN-γ. He detected that administration
36 of IFN-γ increased plasma concentration of ACTH and cortisol but plasma glucose was not altered indicating that IFN-γ is a minor stimulator of endocrine and metabolic pathways (201). However the study did not rule out the effect of IFN-γ in endocrine and metabolic pathways during infection. Some studies revealed that IFN-γ reduced the development of the exoerythrocytic parasite stage and the developing parasitemia which was associated with hypoglycemia (202, 203).
Renal involvement in malaria Renal involvement occurs in both UM and severe malaria. A study carried in Tamale, northern Ghana revealed sub clinical impairment of renal function in 17% of children with UM. Eighty five percent of them had protienuria of either glomerular or tubular pattern indicating that glomerulonephritis and tubular damage can occur in UM (204).
Renal involvement commonly comes in combination with other malaria complication. Children with CM had higher rate of developing acute renal failure and it was usually severe in comparison with children with UM (205). A study Carried in Gambia compared renal function in terms of serum creatinine, pronteinuria and creatinine clearance in patients with cerebral malaria, UM and other febrile illnesses. It revealed that children with CM had higher serum creatinine and higher levels of proteinuria than the other two groups. Children with UM had higher serum creatinine and higher levels of proteinuria than the other febrile illnesses group (206). Renal involvement in malaria presents as acute renal failure, tubulointerstitial nephritis or as nephrotic syndrome. Plasmodium falciparum and plasmodium vivax are responsible for 80.9% and 11.7% of cases of acute renal failure respectively detected by a study conducted
37 in Banaras Hindu University, India during the period 1995-1999 (206). Children were oliguric or anuric in most of the cases, 62% recovered completely and about 18.5% died. Age, oliguria, central nervous system involvement, disseminated intravascular coagulation and multiorgan failure were detected as bad prognostic factors in renal failure due to malaria (207, 208).
Histopathologically, the kidney shows tubular degeneration, endothelial cell swelling and cytoplasic vacuolation. Mononuclear cell with malaria pigment granules in their cytoplasm and sequestration of parasitized RBCs were seen in the glomerular capillaries (209).
Pathogenesis of acute renal failure is multifactorial. In one study hypovolemia was found to be the dominant cause of cases while hyperbilirubinemia, intravascular haemolysis and sepsis were the other causes (207). Sequestration of parasitized red blood cells in the renal vasculature leads to obstruction of the flow and renal ischemia which can result in acute renal failure (209). Rare complication of Plasmodium falciparum infection, myositis, through rhabdomyolysis and myglobinuria was reported to cause acute renal failure (210).
Tubulointerstitial nephritis can present as proteinuria or even acute renal failure. Kidney tubular cells express TNF-α gene and release this cytokine upon stimulation by lipopolysaccharide (211). Thus, the tubular cells have a capacity to modulate immune response within the kidney during inflammation. A study carried on mice attribute pathogenesis of tubulointerstitial nephritis to cytokines dysregulation. High serum TNF- α and IL-10 were reported together with increased expression of TNF-α, IL-1 and IL-6 in both the tubules and the walls of the arteries in the interstitium. There is positive correlation between antibody staining for
38 TNF-α and IL-1 in the tubules and the level of proteinuria. IL-10 staining was found in normal tubules and arteries. There was inverse correlation between staining for IL-10, TNF-α, IL-1 and the degree of proteinuria. These findings implicate TNF-α as a cause of tubular damage which might be due to direct cytotoxic mechanism or through an autoimmune mechanism. IL-10 was thought to be protective as it was only found in normal tubules (212). Electrolyte changes Electrolyte changes are common in malaria. Hyponatremia and hyperkalaemia were encountered on admission while hypokalaemia, hypomagnesemia and hypocalcaemia were detected one day after admission after correction of acidosis (213).
Hypokalaemia & hyperkalaemia: Acidosis is a recognized complication of the severe malaria and a predictor of fatal outcome (214, 215). Alterations in plasma potassium concentrations are commonly associated with acidosis (213). There is little information about the changes in potassium in severe malaria. A prospective study examining the changes in plasma potassium in the first 24 hrs following admission in children with severe malaria presented with impaired consciousness or deep breathing and acidosis, detected that on admission, serum potassium was normal (3.0-5.5 mmol/L) in 81.6%, low (<3 mmol/L) in 11% and high (5.5mmol/L) in 6.3% of children. Plasma potassium decreased rapidly within 4-8 hrs of admission where 40% of patients became hypokalemic after correction of hypovolemia by normal saline infusion. Fractional excretion of potassium and the transtubular gradient of potassium were above the normal range, indicating renal potassium loss. So on correction of acidosis, plasma potassium decreases precipitously
39 and thus careful, serial monitoring of serum potassium is suggested in patients with severe malaria complicated by acidosis (216).
Hyperkalaemia may complicate severe malaria and it is associated with increased mortality. Maitland et al (2005) conducted a study in Kenyan children detected that at admission to the hospital, hyperkalaemia complicated 16% of cases of acidosis due to severe malaria of whom 78% died soon after admission. After admission, mild asymptomatic deficiencies in magnesium and phosphate levels were common but were not associated with any deleterious effect. Asymptomatic potassium deficiency developed despite provision of this electrolyte at maintenance doses (213). These cases were hypoglycemic. Presumably potassium entry with glucose had led to hypokalaemia.
Hyponatremia: Hyponatremia is common in African children with severe malaria, but the cause is unknown. ADH has a controversial role in hyponatremia pathophysiology. ADH secretion is found to be inappropriate in some studies (217, 218) and appropriate to the degree of dehydration in others (219). Sowunmi et al (2000) measured plasma sodium and arginine vasopressin concentrations in 30 children with severe malaria detected hyponatraemia in 53% of the children and was unrelated to peripheral parasite density, dehydration or abnormal renal function. In 67% of children with hyponatraemia plasma ADH levels were inappropriate indicating that ADH secretion is common in children with severe malaria and may influence fluid therapy after correction of initial dehydration (217). Similar finding was reported by Holst et al (1994) (218)
However, Hanson et al (2009) showed that hyponatremia in severe malaria is not caused by inappropriate ADH secretion but reflected
40 hypotonic fluid intake. The study involved 171 Bangladeshi adults with severe malaria. On admission, 57% of patients were hyponatremic. Plasma ADH hormone concentrations were similar in hyponatremic and normonatremic patients and mortality was lower in hyponatremic than normonatremic patients. Plasma sodium normalized with crystalloid rehydration form indicating that the hyponatremia was due to continued oral hypotonic fluid intake in the setting of hypovolemia and requires no therapy beyond rehydration (219). Whether these findings can be extrapolated to children remains uncertain.
Renal impairment during infection is found to result in sodium loss. Renal function was assessed in 40 children during the acute illness and after recovery from falciparum malaria. Creatinine clearance was significantly lower during the acute illness than after recovery. Hyponatraemia occurred in 12.5% during the acute phase and fractional sodium excretion was raised in 27% during the acute illness and continuing sodium wastage occurred in 17% after recovery (220).
Cerebral salt wasting syndrome is another reported mechanism of hyponatremia which occurs in cerebral malaria. In a prospective study, hyponatraemia was observed in 52.6% of children with cerebral malaria on admission and 10% developed hyponatraemia between 48 and 96 hrs after admission. In half of cases, there was continuing urinary sodium loss. An increase in plasma sodium in the first 24 hours of admission was associated with a reduction in seizure frequency during this period as compared to the reported 24 hour of pre-admission seizure frequency (221)
41 Heamoglobinuria
Haemoglobinuria is a severe malaria complication known to occur in long-term residents in Plasmodium falciparum endemic areas who take quinine irregularly and in European expatriates. Rogier et al (2003) conducted a prospective study involved the whole population of a Deilmo, a village in Senegal detected three cases of haemoglobinuria in children who had malaria every 4-6 weeks over many years and repeatedly received quinine (222). This complication became less frequent when chloroquine was the drug of choice for malaria from 1950 until the 1990s (223). As P. falciparum resistance to chloroquine developed, quinine was increasingly used in clinical practice for treating malaria infections. Haemoglobinuria seemed to reappear at the end of the 1990s (224-225). Glucose-6-phosphate dehydrogenase (G6PD) deficiency was also frequently associated with the syndrome (226).
Other antimalarials are also found to be associated with haemoglobinuria. Brunnel et al (2001) reported 21 cases of black water fever in European expatriates who lived in sub-Saharan Africa. The presumed triggers of black water fever were halofantrine, quinine, mefloquine and halofantrine or quinine (227).
Clinical features characterized by severe intravascular haemolysis and anaemia producing dark urine in patients with severe malaria. Abdominal pain, jaundice, hepatosplenomegaly, vomiting, and renal failure have also been reported (226).
The pathogenesis of BWF remains unclear (222, 227). Its management changed with the introduction of artemisinin derivates but is still debated. Price et al (1999) state that parenteral quinine can be stopped when
42 artemisinin derivatives are available because they seem to be safe and well tolerated (228).
43 Rational of the study Malaria continues to claim one to three million lives a year mainly those of children in sub Saharan Africa. A study in Gambia reported 80% deaths to occur during the first day of admission and childhood mortality remains unacceptably high (9). In the Sudan, it is responsible for 44.000 deaths annually (5). Despite the huge literature, the pathophysiological mechanisms of severe malaria are still not well known and better understanding of these mechanisms and their application may open a window for discovery of new therapeutic agents and prevention methods. Identifying bad prognostic factors has great advantages in aiding clinicians in identification of sickest children who need special care as well as targeting resources to children at higher risk.
This project investigates the endocrine and paracrine aspects of the pathophysiology of severe malaria in children up to 12 years. This study monitors the clinical and laboratory parameters during the early stages of severe malaria so as to identify and correct life threatening changes. The study also aims to identify indicators of high risk patients.
44 OBJECTIVES
General objectives: To monitor clinical manifestations and selected laboratory tests in children with severe malaria so as to identify indicators of serious outcomes of the disease.
Specific objectives: 1. In addition to a comprehensive history, physical examination and clinical follow up, the study has focused on the following battery of tests: a- Blood glucose homeostasis including insulin and C-peptide assay. b- Cytokines (TNF-α, IFN-γ and IL-10) c- Haemoglobin and total white blood cells estimation d- Hepatic functions e- Renal functions f- Serum electrolytes 2- Based on the above, the study aims at recommendation which may lead to a cost effective protocol of appropriate laboratory investigations and clinical monitoring 3- Indicators to identify high risk patients would be an essential part of a protocol for management of severe malaria in children.
45 CHAPTER TWO PATIENTS AND METHODS Location and time of the study The study was conducted at Wadmedani Paediatric Hospital. The collection of samples started in September 2007 and extended up to September 2008. Patients and controls The study included a cohort of fifty six children with severe malaria who reported to the hospital within the above period and were included according to WHO criteria of SM. Thirty one children with uncomplicated malaria were included in the study for comparison. The age of both groups ranged from 2 year up to 12 years. Exclusion criteria identified patients who had antimalarial treatment before admission, those known to have renal disease, hepatic disease, diabetes mellitus or those suffering from metabolic disorders as well as malnourished children (based on weight for age charts). The study was approved by the Institutional Ethical Committee of University of Khartoum and verbal consent was obtained from parents of the children. History and physical examination Both groups of patients underwent a history and comprehensive physical examination by the researcher as well as by hospital staff. A data collection form was completed for each patient including history, physical examination and laboratory findings. Malaria diagnosis and blood tests: Blood samples A sample consisting of 5 ml was taken from each patient on admission and before treatment and divided into 3 tubes, one containing EDTA used for Hb, TWBCs count and parasitemia estimation. Second tube containing fluoride was used for RBG and the third one was a plain tube
46 for the rest of investigations. For each patient thick and thin blood film, Hb estimation, TWBCs count, RBG estimation, insulin and C-peptide measurement, urea, creatinine, electrolytes sodium & potassium, total bilirubin and differential, liver enzymes; AST, ALT and ALP and Cytokines; TNF-α, IFN-γ and IL-10 were done. Malaria diagnosis & parasitemia estimation The microscopic identification of the malaria parasite is the method of confirmation of infection with plasmodium falciparum used in the study to satisfy WHO criteria. Only positive cases were included. The thick blood film was used for parasite detection while thin film was used for identification of species and parasitemia estimation. The thin blood film was fixed using absolute methyl alcohol, and then stock solution of Giemsa stain was diluted with distilled water. The distilled water was kept at pH 7.1-7.2 using phosphate buffer. Staining of films was done. Parasitemia estimated in thin films expressed as a percentage of infected RBCs from total RBCs (229). Haemoglobin (Hb) estimation Haemoglobin was measured by a colorimetric method. Hb was oxidized by potassium ferricyanide to methaemoglobin which is in turn converted to cyanomethaemoglobin by potassium cyanide. The intensity of color produced was proportional to Hb concentration in the sample (230). Procedure of test started by preparation of working reagent by mixing 1 vial of drabkin solution with 245ml of distilled water. Then a set of three tubes were prepared. In the blank tube, 5ml of the working reagent was added. Into the standard tubes 5ml of the working reagent and 20µl of Hb standard were added. Into the sample tubes, 5ml of working reagent and 20µl of sample were added. Mixing and incubation for three minutes at room temperature was done. Absorbance of the sample and standard was measured at 540nm against the blank.
47 Total white blood cells (TWBCs) count TWBCs were counted in haemocytometer after mixing of 0.02ml of blood with 0.38ml of glacial acetic acid (231). Random blood glucose measurement RBG was measured by enzymatic colorimetric method. The glucose was oxidized to gluconate in the presence of glucose oxidase with formation of hydrogen peroxide. Hydrogen peroxide oxidizes the mixture of phenol and 4-aminoantipyrine to red quinoneimine dye proportional to concentration of the glucose present in the sample (232). The test began with preparation of a set of three tubes then 1ml of reagent was added to each of the three tubes. The first tube is considered the blank, to second tube 10µl of sample was added and labeled as sample tube and to the third tube 10µl of standard was added and labeled as standard tube. The three tubes were incubated for 10 minutes at room temperature, and then absorbance of standard and sample were road at 500 nm against the reagent blank. Hormone assays Insulin and C-peptide measurement Insulin and C-peptide concentrations were measured in the serum using radioimmunoassay. Principle of radioimmunoassay The radioimmunoassay in this test depends upon the competition iodine- 125 labeled analyte (insulin and C-peptide) and the analyte in the patient sample or in the standard for the limited number of binding site on the analyte specific antibody. After incubation for a fixed time, separation of the bound from free is achieved by polyethylen glycol-accelerated double antibody procedure. The tube then counted in a gamma counter, the counts being inversely related to the amount of analyte present in the patient sample. By measuring the proportion of iodine-125 labeled
48 analyte bound in the presence of varying known amount of analyte standards, the concentration of the analyte in unknown samples can be interpolated (233). Procedure of insulin assay Procedure of insulin assay started by labeling and arrangement of test tubes according to the scheme protocol of the kit producers then 100µl of the insulin standards solution and the unknown samples were pipetted into the appropriate test tubes. This step was followed by addition of 100µl of insulin antibody solution to all tubes except total and non specific binding tubes. Then 100µl of iodine-125 labeled insulin solution were added to all tubes except the blanks and all test tubes were incubated at 37ºC for 2 hours. 500µl of the separating solution were added to all tubes except total count tube and the tubes were mixed and incubated at 37ºC for 15 minutes. All test tubes were centrifuged (except total count tube) at 1500 rpm for 15 minutes and the supernatant of all tubes were decanted. Finally all the tubes were counted in a gamma counter. Procedure of C-peptide assay Test tubes were labeled and arranged according the scheme protocol then 100µl of the C-peptide standards solution and the unknown samples were pipetted into the appropriate tubes. 100µl of C-peptide antibody solution were added to all tubes except total and non specific binding tubes followed by addition of 100µl of iodine-125 labeled C-peptide solution and incubation of all test tubes at 4ºC for 24 hours. Then 500µl of the separating solution were added to all tubes except total count tube, then the tubes were mixed and incubated at 37ºC for one hour. The supernatant of all tubes were decanted and all the tubes were counted in a gamma counter.
49 Urea measurement Urea is measured by a UV enzymatic method using spectrophotometer. Urea was hydrolyzed by urease to ammonia and carbon dioxide. The ammonia is then converted to glutamate by glutamate dehydrogenase in the presence of oxoglutarate and NADH. The reaction is then monitored kinetically at 340nm by the rate of decrease in absorbance resulting from the oxidation of NADH to NAD+ proportional to concentration of urea present in the sample (234). Procedure of the test started by preparation of the working reagent by mixing 4ml of reagent one which contain urease enzyme with 1ml of reagent two which is the buffered chromogen. The working reagent, standard and samples were incubated at 37°C. One ml of working reagent was added to set of two tubes, one containing 10µl of sample and the other containing 10µl of standard. Inversion of both tubes several times was done. The spectrophotometer was set to zero absorbance by distilled water. The absorbance of both tubes was measured at 340nm at different fixed time and then the concentration of urea was estimated. Creatinine measurement Creatinine was measured by a kinetic method. Creatinine under alkaline condition reacts with picrate ions forming a reddish complex. The formation rate of complex measured through the increase in absorbance in a prefixed interval of time is proportional to the concentration of creatinine in the sample (235). Procedure of the test started by preparation of the working reagent by mixing one volume of reagent one (picric acid) with one volume of reagent two (alkaline buffer). The working reagent, standard and samples were incubated at 37°C. One ml of working reagent was added to set of two tubes, one containing 100µl of sample and the other containing 100µl of standard. Mixing of each tube was done. The spectrophotometer was
50 set to zero absorbance by distilled water. The absorbance of both tubes was measured at 510nm at different fixed time and then the concentration of creatinine was estimated. Sodium and potassium measurement Sodium and potassium were measured in serum samples using a flame photometer (236). Procedure of the test involved addition of 0.1 ml of sample to 9.9 ml of distilled water in a test tube before measurement in the flame photometer. Bilirubin measurement Bilirubin was measured using a colorimetric method. Bilirubin was converted to color azobilirubin by sulfanilic acid and measured photometrically. Bilirubin glucuronide reacts directly in aqueous solution while indirect bilirubin requires solubilization with dimethylsulfoxide to react (237). Procedure of determination of total bilirubin started with prepation of the working reagent by mixing 4ml of reagent totat (RT) and 1ml of sodium nitrate. Then a set of four tubes was prepared. Into sample blank tube 100µl of sample and 1ml of RT were added. In the sample tubes, 100µl of sample, 1 ml of working reagent were added. In the calibrator blank 100µl of distilled water, 1ml of working reagent were added. Into the calibrator tube 100µl of calibrator and 1ml of working reagent were added. Then the tubes were mixed thoroughly and allowed to stay for two minutes at room temperature. The absorbance of sample blank was measured at 540nm against distilled water and that of sample against calibrator blank. For determination of direct bilirubin, working reagent was prepared by mixing 4ml of reagent direct (RD) and 1ml of sodium nitrate. Then a set of four tubes was prepared. Into sample blank tube 100µl of sample and 1ml of RD were added. In the sample tubes, 100µl of sample, 1 ml of
51 working reagent were added. In the calibrator blank 100µl of distilled water, 1ml of working reagent were added. Into the calibrator tube 100µl of calibrator and 1ml of working reagent were added. Then the tubes were mixed thoroughly and allowed to stay for two minutes at room temperature. The absorbance of sample blank was measured at 540nm against distilled water and that of sample against calibrator blank. Alanine aminotransferase (ALT) measurement ALT was measured by UV enzymatic method. ALT catalyzes the transfer of amino group from alanine to oxoglutarate with the formation of glutamate and pyruvate. The latter was reduced to lactate by adding lactate dehydrogenase in the presence of NADH. The reaction was monitored kinetically at 340nm by the rate of decrease in absorbance resulting from the oxidation of NADH to NAD+ which is proportional to the activity of ALT present in the sample (238). Procedure of the test started by preparation of the working reagent by mixing 4ml of reagent one (ALT substrate) with 1ml of reagent two (ALT coenzyme). Then working reagent, standard and samples were incubated at 37°C. One ml of working reagent was added to set of two tubes, one containing 50µl of sample and the other containing 50µl of standard. Mixing by inversion of both tubes several times was done. The spectrophotometer was set to zero absorbance by distilled water. The absorbance of both tubes was measured at 340nm at different fixed time and then the concentration of ALT was estimated. Aspartate aminotransferase (AST) measurement AST was measured by a UV enzymatic method. AST catalyzes the transfer of amino group from aspartate to oxoglutarate with the formation of glutamate and oxaloacetate. The latter is reduced to malate by malate dehydrogenase in the presence of NADH. The reaction was monitored kinetically at 340nm by the rate of decrease in absorbance resulting from
52 the oxidation of NADH to NAD+ which is proportional to the activity of AST present in the sample (239). Procedure of the test began with preparation of the working reagent by mixing 4ml of reagent one (AST substrate) with 1ml of reagent two (AST coenzyme). Working reagent, standard and samples were incubated at 37°C. One ml of working reagent was added to set of two tubes, one containing 50µl of sample and the other containing 50µl of standard. Mixing was achieved by inversion of both tubes several times. The spectrophotometer was set to zero absorbance by distilled water. The absorbance of both tubes was measured at 340nm at different fixed time and then the concentration of ALT was estimated. Alkaline phosphatase (ALP) measurement ALP was measured by colorimetric method. ALP catalyzes the hydrolysis of 4-nitrophenyphosphate to 4- nitrophenol and inorganic phosphate. The reaction was monitored kinetically at 405nm by the rate of formation of 4-nitrophenol which is proportional to the activity of ALP present in the sample (240). Procedure of the test started by preparation of the working reagent by mixing 4ml of reagent one (ALP buffer) with 1ml of reagent two (ALP substrate). Then working reagent, standard and samples were incubated at 37°C. One ml of working reagent was added to set of two tubes, one containing 20µl of sample and the other containing 20µl of standard. Inversion of both tubes several times was done. The spectrophotometer was set to zero absorbance by distilled water. The absorbance of both tubes was measured at 405nm at different fixed time and then the concentration of ALT was estimated.
53 Cytokines measurement Measurement of serum cytokines was done using ELISA technique. ELISA principle The assay is based on a blend of antibodies directed against the cytokine. These antibodies are fixed to the surface of the wells. Standards or samples, containing the cytokine, react with the antibodies coated in the well and with antibodies labeled with enzyme. After the incubation period at predetermined time allowing the formation of sandwich, the plate is washed to remove unbound enzyme labeled antibodies. Then, substrate solution is added and incubated. The reaction is stopped by stop solution. The plate is then read at appropriate wavelength. The amount of substrate turnover is determined colorimetrically by measuring the absorbance which is proportional concentration. Standard curve is plotted and cytokine concentration is determined by interpolation from the standard curve (241). The procedure followed were those recommended by kit manufacturer. Procedure of IFN-γ assay The assay started by addition of 50µl of each standard, control and samples to appropriate well then 50µl of anti- IFN-γ conjugate were added into all the wells. Incubation for 2 hours at room temperature on a horizontal shaker set at 700 rpm± 100 rpm was allowed and followed by washing of the plate using an automatic washer. Then 200µl of freshly prepared chromogenic solution were added into all the wells followed by incubation for 15 minutes at room temperature on a horizontal shaker set at 700 rpm ± 100 rpm. The reaction was stoped by addition of 50µl of stop solution for each well. Absorbance was read at 450nm and 490 and the standard curve was plotted using optical density against standard concentrations.
54 Procedure of TNF-α assay At the beginning of assay, 50µl of standard, samples and sample diluent were added into appropriate wells then the test tubes were incubated for 1hour at room temperature. Washing was done 5 times followed by addition of 50µl of biotin antibody into each well and the tubes again incubated for 30 minutes at room temperature. Another washing was done 5 times followed by addition of 50µl of HRP-streptavidin solution into each well. Incubation for 30 minutes at room temperature was allowed and followed by washing 5 times. Then 50µl of TMB substrate solution into were added to each well followed by incubation for 20 minutes at room temperature and addition of 25µl of stop solution. Absorbance was read at 450nm against 630nm Procedure of IL-10 assay Procedure of IL-10 assay was started by addition of 50µl of standard, samples and sample diluent into appropriate wells then 50µl of biotin antibody were added into each well. Incubation for 1 hour 30 minutes at room temperature was allowed followed by washing 5 times. Then 1ooµl of HRP-streptavidin solution was added into each well and the wells were incubated for 30 minutes at room temperature which was followed by washing 5 times. Then 50µl of TMB substrate solution into each well and incubation for 20 minutes at room temperature was allowed. This step was followed by addition of 25µl of stop solution into each well. Absorbance was read at 450nm against 630 nm. Statistical methods: Statistical analysis was carried out with stastical package of social sciences (SPSS) for windows version 11.5. SPSS version 15 was used to display correlation graphs. Normality of data distribution was checked using Kolmogrov-Smirnov test. Data were presented as means and standard deviations and analyzed by Student's T test. Correlations were
55 compared using the χ2 tests if the variables were qualitative, Pearson's correlation coefficient was used for quantitive variables and Spearman' rank correlation coefficient tests was used to correlate quantitive variable with qualitive variable. Assessments of prognostic factors were conducted with regression model. P values < 0.05 were considered as significant.
56 CHAPTER THREE RESULTS Sample characteristics The total number of patients with malaria included in the study was 87 patients. WHO guidelines for severe malaria were used to divide the patients into two categories; uncomplicated malaria (UM) and severe malaria (SM) cases. Sample characteristics are shown in the table below Table-1 Sample characteristics Character UM SM P value Number 31 56 Age(months) 82.71( 26-144) 56.75(24- 0.001 mean &range 144) Gender 0.458 Male 17(54.8%) 26(46.4%) female 14(45.2%) 30(53.6) Shaded box indicate significance. Figure-1 Age distribution of UM and SM
100
80
60
40
age classes 20
Lless than 5 years
0 more than 5 Percent uncomplicated malari severe malaria
presentation
Severe malaria affects younger children while uncomplicated malaria affects older children (figure-1). Significant difference was found
57 between the two means (P value=0.001). In comparing the means of different severe malaria categories, children with severe anaemia and repeated convulsions were younger than those with cerebral malaria. Haemoglobinuric patients were significantly older than those without haemoglobinuria (P value=0.007). There was a significant association between the age of the child and presenting category of malaria (P value=0.000). Clinical presentation Fever, nausea and vomiting were the most common symptoms of children having uncomplicated malaria. Jaundice and hepatosplenomegaly were infrequent in this group. In children with severe malaria fever and repeated convulsions were the most frequent presenting features. Table-2 The clinical presentation of children with malaria Clinical feature UM- UM-percentage SM- SM- number number percentage Fever 31 100 56 100 Cough 11 35.5 17 30.4 Shortness of 0 0 8 14.3 breath Nausea 19 61.3 21 37.5 Vomiting 19 61.3 21 37.5 Diarrhea 10 32.3 10 17.9 Convulsions 2 6.5 23 41.1 Tackycardia 14 45.2 47 83.9 Tackypnoea 10 32.3 40 71.4 Jaundice 1 3.2 8 14.3 Hepatomegaly 2 6.5 11 19.6 Splenomegaly 0 0 12 21.4 Cerebral malaria - 13 23.2 Severe anaemia - 13 23.2 Hypoglycemia - 1 1.8 Haemoglobinuria - 2 3.6 Mixed - 4 7.1 presentation
58 Table-3: Laboratory investigation of UM & SM Investigation Kolmogro UM SM P v-Smirnov (mean± SD) ( mean ±SD) value test P value RBG(mg/dl) 0.367 108±39.14 89.27±39.06 0.035 Urea(mg/dl) 0.076 22.784±6.14 25.14±14.72 0.398 Creatinine(mg/dl) 0.063 0.561±0.22 0.40±0.32 0.016 Na+(mmol/l) 0.200 135.03±3.30 133.20±4.65 0.056 K+(mmol/l) 0.067 3.84±0.41 3.98±1.04 0.474 AST(U/L) 0.271 34.61±23.09 26.96±20.83 0.118 ALT(U/L) 0.203 22.48±15.44 19.86±12.96 0.401 AIP(U/L) 0.697 400.48±120.95 398.89±144.3 0.959 1 Total 0.575 0.681±0.28 1.130±0.99 0.016 bilirubin(mg/dl) Hb(g/dl) 0.871 9.91±1.67 7.54±2.71 0.000 TWBCs(cell/µl) 0.566 5419.35±1661. 8169.64±553 0.009 61 6.26 Shaded boxes indicate significance. Each two means were compared using the Student’s T test. Blood glucose The mean blood glucose level in uncomplicated malaria was 108.06mg/dl while severe malaria group had mean blood glucose of 89.27mg/dl. The difference between the two means was significant (P value =0.035). Hypoglycemic patients and those with mixed presentation had the lowest blood glucose and showed significant differences when compared to children with uncomplicated malaria.
59 Table-4 Comparison of RBG between UM&SM categories UM (mean±SD) SM categories Number Mean ±SD Pvalue
108.06±39.14 Cerebral malaria 13 95.54±30.82 0.311 Severe anaemia 13 79.92±35.91 0.031 Repeated 23 100.17±39.06 0.467 convulsions Hypoglycemia 1 22.00 0.039 Haemoglobinuria 2 94.00±43.84 0.627 Mixed 4 51.00±47.51 0.011 presentation Shaded boxes indicate significance. Insulin The mean insulin level in UM was 8.35mIU/L while in SM was 8.87mIUl/L. No significant difference was found (P value=0.754). Three cases of hyperinsulinemia were found in the severe malaria group. No significant differences were found between clinical categories of severe malaria and UM. Table-5 Comparison of insulin between UM & SM categories UM(mean±SD) SM categories Number Mean ±SD P value 8.35±3.01 Cerebral malaria 12 11.29±14.57 0.286 Severe anaemia 12 7.25±1.74 0.241 Repeated 23 9.18±8.59 0.622 convulsions Hypoglycemia 1 6.91 0.639 Haemoglobinuria 2 7.39±0.81 0.658 Mixed 4 5.94±1.07 0.125 presentation
C-peptide The mean of C-peptide in UM was 1.04ng/ml while in SM 1.88ng/ml. No significant difference was found between the two (P value=0.172). Children with severe anaemia and mixed presentation had significantly higher C-peptide level than those with UM.
60 Table-6 Comparison between C-peptide in UM & SM categories UM (mean±SD) SM categories Number Mean ±SD P value 1.04±0.65 Cerebral malaria 13 1.99±2.94 0.099 Severe anaemia 10 3.31±6.07 0.045 Repeated 22 0.92±0.46 0.452 convulsions Hypoglycemia 1 0.53 0.448 Haemoglobinuria 2 0.87±0.25 0.725 Mixed 4 4.15±4.17 0.000 presentation Shaded boxes indicate significance. Insulin: C-peptide ratio The ratio of Insulin: C-peptide ratio in UM was 0.22 while in SM 0.18. No significant difference was found in between two (P value=0.074). Children with mixed presentation had significantly lower ratio than UM.
Figure-2 Ratio of insulin to C-peptide in UM & S
.23
.22
.21
.20
.19
.18
.17 Mean ratio I/C UM SM
presentation
61 Table-7 Comparison between insulin: C-peptide ratio in UM & SM categories UM (mean±SD) SM categories Number Mean ±SD Pvalue
0.22±0.11 Cerebral malaria 12 0.15±0.07 0.066 Severe anaemia 9 0.16±0.11 0.192 Repeated 22 0.21±0.11 0.628 convulsions Hypoglycemia 1 0.28 0.629 Haemoglobinuria 2 0.18±0.03 0.634 Mixed 4 0.08±0.09 0.022 presentation Shaded box indicate significance. Correlation between insulin and blood glucose Significant positive correlation was found between insulin and blood glucose (P value= 0.006) using Pearson's correlation coefficient.
Figure-3 Correlation between blood glucose and insulin
62 Correlation between insulin and C-peptide Significant positive correlation was found between insulin and C-peptide (P value=0.000) using Pearson's correlation coefficient.
Figure-4 Correlation between insulin and C-peptide
Table-8 Expected value of insulin to C-peptide ratio in UM & SM Parameter UM SM Recorded insulin 50.14 53.24 pmol/l C-peptide pmol/l 290.23 524.66 Expected value of 58.04 104.93 insulin
Haemoglobin The mean Hb level in uncomplicated malaria was 9.91g/dl while severe malaria group had mean Hb of 7.54g/dl. A significant difference was found between the two means (P value=0.000). Children with severe
63 anaemia and mixed presentation as well as those with cerebral malaria had the lowest Hb levels and significant differences were found between their means and uncomplicated malaria.
Table-9 Comparison of Hb between UM & SM categories UM ( g/dl) SM categories Number Mean ±SD P value 9.91±1.67 Cerebral 13 8.39±1.55 0.008 malaria Severe anaemia 13 4.05±0.77 0.000 Repeated 23 8.93±2.03 0.060 convulsions Hypoglycemia 1 7.70 0.204 Haemglobinuria 2 10.90±2.68 0.435 Mixed 4 6.40±3.27 0.001 presentation Shaded boxes indicate significance. Total white blood cells count The mean total white blood cells count of uncomplicated malaria was 5419.35 cells/ µl while in severe malaria 8169.64 cells/µl. A significant difference was found between the two means (P value=0.009). Children with mixed presentation, cerebral malaria and repeated convulsions had the highest TWBCs count
Table-10 Comparison of TWBCs between UM & SM categories UM SM categories Number Mean ±SD P value 5419.35+1661.61 Cerebral malaria 13 8307.69±3635.03 0.001 Severe anaemia 13 6092.31±2986.77 0.34 Repeated 23 7465.22±3488.76 0.006 convulsions Hypoglycemia 1 7500 0.227 Haemoglobinuria 2 6100±141.42 .5720 Mixed 4 19725± 13433.63 0.000 presentation Shaded boxes indicate significance.
64 Bilirubin The mean bilirubin level in uncomplicated malaria was 0.68 mg/dl and in severe malaria 1.13mg/dl. A significant difference was found between the two means (P value=0.016). Children with mixed presentation, cerebral malaria and repeated convulsions had the highest total bilirubin level which was mainly of the unconjugated form. A significant correlation was found between bilirubin and urea (P value=0.000) and between bilirubin and creatinine (P value=0.001) using Pearson's correlation coefficient. Table- 11 Comparison of bilirubin between UM & SM UM SM categories Number Mean ±SD P value 0.68+0.28 Cerebral malaria 13 1.16±0.68 0.002 Severe anaemia 13 0.89±0.61 0.125 Repeated 23 1.06±0.76 0.013 convulsions Hypoglycemia 1 0.20 0.112 Haemoglobinuria 2 0.60±0.00 0.700 Mixed 4 2.67±2.53 0.000 presentation Shaded boxes indicate significance. Liver enzymes No significant differences were found between liver enzymes of uncomplicated and severe malaria as well as in comparing uncomplicated malaria with different severe malaria categories, however children with severe malaria had a significantly higher frequency of high levels of these enzymes when compared to uncomplicated malaria using χ2 test for goodness of fit. Table -12 Mean of AST. ALT & ALP in UM & SM Enzyme UM SM P value AST 34.61±23.09 26.96±20.83 0.118 ALT 22.48±15.48 19.86±12.96 0.401 ALP 400.48±120.95 398.89±144.31 0.959
65 Table-13 Comparison of AST between UM & SM UM SM categories Number Mean ±SD P value 34.61±23.09 Cerebral malaria 13 38.92±28.61 0.602 Severe anaemia 13 21.08±16.03 0.061 Repeated 23 26.83±17.68 0.183 convulsions Hypoglycemia 1 6.00 0.232 Haemoglobinuria 2 10.00±5.65 0.148 Mixed 4 21.75±14.38 0.288 presentation
Figure-5 AST in UM & SM
120
100
80
60
40
20
0 AST 0 1 2 3
presentation
1=uncomplicated malaria 2=severe malaria
66 Table-14 Comparison of ALT between UM & SM categories UM SM categories Number Mean ±SD P value 22.48±23.09 Cerebral malaria 13 19.85±14.14 0.602 Severe anaemia 13 19.62±11.87 0.061 Repeated 23 21.17±13.58 0.183 convulsions Hypoglycemia 1 2.00 0.232 Haemoglobinuria 2 11.50±9.19 0.148 Mixed 4 21.75±12.86 0.288 presentation
Figure-6 ALT in UM & SM
80
60
40
20
0 ALT 0 1 2 3
presentation
1=uncomplicated malaria 2=severe malaria
67 Table-15 Comparison of ALP between UM & SM categories UM SM categories Number Mean ±SD P value 400.48±120.95 Cerebral malaria 13 368.23±170.23 0.480 Severe anaemia 13 401.08±151.00 0.989 Repeated 23 389.74±123.48 0.750 convulsions Hpoglycemia 1 700.00 0.021 Haemoglobinuria 2 373.50±178.89 0.766 Mixed 4 481.50±87.93 0.206 presentation
Figure-7 ALP in UM&SM
800
700
600
500
400
300
200
100
0 ALP 0 1 2 3
presentation
1=uncomplicated malaria 2=severe malaria Urea The mean urea level in uncomplicated malaria was 22.78mg/dl while in severe malaria 25.14mg/dl. No significant difference was found between the two (P value=0.398). In comparing uncomplicated malaria with
68 different severe malaria categories, significant difference was found with mixed presentation (P value=0.000).
Table-16 Comparison of urea between UM & SM categories UM SM categories Number Mean ±SD P value 22.78±6.14 Cerebral malaria 13 27.4±8.71 0.052 Severe anaemia 13 21.35±8.59 0.536 Repeated 23 21.74±7.30 0.573 convulsions hpoglycemia 1 16.00 0.286 haemoglobinuria 2 19.00±4.24 0.401 Mixed 4 55.00±39.54 0.000 presentation Shaded box indicate significance. Creatinine The mean creatinine level in uncomplicated malaria was 0.56mg/dl while in severe malaria 0.40mg/dl. A significant difference was found between the two (P value=0.016). Children with haemoglobinuria, hypoglycemia, repeated convulsions and severe anaemia showed significant differences in comparison with uncomplicated malaria.
Table-17 Comparison of creatinine between UM& SM categories UM SM categories Number Mean ±SD P value 0.56±0.22 Cerebral malaria 13 0.41±0.21 0.054 Severe anaemia 13 0.36±0.22 0.011 Repeated 23 0.36±0.15 0.001 convulsions Hypoglycemia 1 0.10 0.052 Haemoglobinuria 2 0.20±.00 0.032 Mixed 4 0.90±.94 0.082 presentation Shaded boxes indicate significance.
69 Sodium The mean sodium level in uncomplicated malaria was 135mmol/l while in severe malaria 133mmol/l no significant difference was found between the two. Children with severe anaemia and repeated convulsions had significantly lower sodium level in comparison with uncomplicated malaria. However, a total of fifty children were found to be hyponatremic, most of them had severe malaria. Incidence of hyponatremia was more frequent in severe malaria than uncomplicated malaria (P value=0.005) using χ2 tests for goodness of fit.
Figure-8 Frequency of hyponatremia in SM
sodium
135-155
less than135
70 Table-18 Comparison of sodium between UM & SM categories UM SM categories Number Mean ±SD P value 135.03±3.30 Cerebral malaria 13 133.31±5.52 0.206 Severe anaemia 13 132.38±3.94 0.027 Repeated 23 132.65±4.42 0.028 convulsions Hypoglycemia 1 140 0.149 Haemoglobinuria 2 134.00±8.48 0.696 Mixed 4 136.50 ±3.00 0.405 presentation Shaded boxes indicate significance Potassium Although the mean potassium level in uncomplicated malaria showed no significant difference compared with severe malaria, hypokalaemia was found to be significantly more frequent in severe malaria than uncomplicated malaria (P value=0.039) using χ2 test for goodness of fit. Those children who had hyperkalaemia presented mostly with neurological manifestation in the form of cerebral malaria or repeated convulsions and one of them died.
71 Figure-9 Incidence of hypokalaemia and hyperkalaemia in UM & SM
40
30
20
potassium
10 less than 3.5
3.5-5.5
0 more than 5.5 Count UM SM
presentation
Table-19 Comparison of potassium between UM & SM categories UM SM categories Number Mean ±SD P value 3.84±0.41 Cerebral malaria 13 4.02±1.38 0.514 Severe anaemia 13 3.97±.80 0.477 Repeated 23 4.09±1.02 0.232 convulsions Hypoglycemia 1 4.20 0.405 Haemoglobinuria 2 3.99±.80 0.455 Mixed 4 3.70±.94 0.578 presentation
Association between the course of malaria and the clinical categories There is significant association between the clinical categories of malaria and course of the disease in the form of length of hospital stay and survival (P value=0.000) using Spearman' rank correlation coefficient test.
72 Table-20 Clinical categories of malaria and outcome Clinical Number Short hospital Increase Death categories of stay(≤2 days) morbidity(>2days) malaria Uncomplicated 31 31(100%) 0 0 malaria Severe malaria 56 51(91%) 4 (7.2%) 1(1.8%) Cerebral 13 11(84.61%) 1 (7.69%) 1(7.69%) malaria Mixed 4 1 (25%) 3 (75%) 0 presenentation
Mixed presentation Children with mixed presentation were found to have low RBG and haemoglobin in comparison with children who had uncomplicated malaria. Also, they had high total white blood cells count, bilirubin and urea. Table-21 The RBG, Hb, bilirubin, TWBCs and urea of uncomplicated malaria and mixed presentation Investigation Uncomplicated Mixed P value malaria ( presentatiom mean ±SD) ( mean ±SD) RBG 108±39.14 51±47.51 0.011 Hb 9.910±1.67 6.4±3.27 0.001 Bilirubin 0.68±0.28 2.675±2.53 0.000 TWBCs 5419.35±1661.61 19725±13433.63 0.000 Urea 22.78±6.14 55.000±39.54 0.000 K 3.84±0.41 3.700±.94 0.578 Shaded boxes indicate significance. Cerebral malaria Children with cerebral malaria were found to have high total white blood cells count and bilirubin and low haemoglobin in comparison with children with uncomplicated malaria.
73 Table-22 The Hb, bilirubin and TWBCs of uncomplicated malaria and cerebral malaria Investigation Uncomplicated Cerebral malaria P value malaria Hb 9.91±1.67 8.39±1.55 0. 008 Bilirubin 0.68±.28 1.16±0.68 0.002 TWBCs 5419.35±1661.61 8307.69±3635.03 0.001 Shaded boxes indicate significance. Parasitemia About 30.4% of children with severe malaria were found to have hyperparasitemia while 19.4% of those with uncomplicated malaria had hyperparasitemia. The incidence of hyperparasitemia was found to more in severe malaria than in uncomplicated malaria (P value=0.022) using χ2 test for goodness of fit. Negative correlation were detected between parasitemia and Hb (P value=0.004) using Pearson's correlation coefficient test. Also significant negative correlation were found between parasitemia and RBG (P value=0.017) using Pearson's correlation coefficient test. No significant correlation were found between the degree of parasitemia and clinical categories of malaria (P value=0.214) using Spearman' rank correlation coefficient as well as with potassium (P value=0.298), sodium (P value=0.244), TNF-α (P value=0.388), IL-10 (P value=0.072) and IFN-γ (P value=0.146). The later five correlations were analyzed by Pearson's correlation coefficient test.
74 Figure-10 Degree of parasitemia in UM & SM
50
40
30
20
parasitemia 10
less or equal 5%
0 more than 5% Count UM SM
presentation
75 Figure-11 Correlation between parasitemia and haemoglobin
parasitemia
76 Figure-12 Correlation between parasitemia and blood glucose
Period of illness The mean period of illness (days of illness before admission) in UM was 2.13 days while in SM was 2.41 days. No significant difference was found (P value=0. 476). Children with severe anaemia had a significantly prolonged period of illness in comparison to UM (P value=0.001). Significant negative correlation was found between period of illness and Hb (P value=0.001), RBG (P value=0. 041) and IFN-γ (P value=0.001) using Pearson's correlation coefficient test. .
77 Table-23 Comparison of period of illness between UM & SM UM SM categories Number Mean ±SD P value 2.13±1.60 Cerebral malaria 13 2.23±1.30 0.841 Severe anaemia 13 4.46±2.57 0.001 Repeated 23 1.61±0.65 0.150 convulsions Hypoglycemia 1 2 0.938 Haemoglobinuria 2 2±0.000 0.912 Mixed 4 1.25±0.50 0.290 presentation Shaded box indicate significance TNF-α The mean of TNF-α of uncomplicated malaria was 238.91pg/ml while in severe malaria was 178.52pg/ml no significant difference was found between the two (P value=0.675). No significant differences were found between severe malaria categories and uncomplicated malaria however children with cerebral malaria had a significantly higher TNF-α than those without cerebral malaria (P value=0.035). Significant difference was found between cerebral malaria and severe anaemia (P value=0.029) Positive correlation between TNF-α and RBG was noted but it wasnot reach significance in both UM and SM (P value=0.798 and 0.172 respectively). Multiple regression models revealed a significant relation between TNF-α and ALT and ALP. TNF-α positively correlated with ALT (P value=0.014) when they analyzed by Pearson's correlation coefficient test.
78 Figure-13 TNF-α in children with cerebral malaria and those without cerebral malaria
280
250
220
190
160 Mean pg/ml TNF-alpha CM w ithout CM
CM
79 Figure-14 TNF-α in different malaria cateories
Table-24 Comparison of TNF-α in UM & SM categories. UM SM categories Number Mean ±SD P value 238.91±725.20 Cerebral malaria 13 258.66±574.81 0.931 Severe anaemia 13 39.70±61.43 0.332 Repeated 23 246.92±813.94 0.970 convulsions Hypoglycemia 1 0.00 0.748 Haemoglobinuria 2 82.70±49.35 0.766 Mixed 4 68.40±61.82 0.646 presentation
80 Table-25 Correlation of TNF-α with clinical and laboratory parameters Parameter P value Age 0.259 Sex 0.385 Hepatomegaly 0.082 Splenomegaly 0.182 Period of illness 0.564 Temperature 0.912 Parasitemia 0.388 Hb 0.562 RBG 0.421 Insulin 0.650 C-peptide 0.466 TWBCs 0.225 Total bilirubin 0.578 Direct bilirubin 0.713 Indirect bilirubin 0.575 AST 0.480 ALT 0.014 ALP 0.252 Urea 0.949 Creatinine 0.704 Sodium 0.751 Potassium 0.632 IL-10 0.879 IFN-γ 0.161 Outcome 0.152 Shaded box indicate significance IFN-γ The mean of IFN-γ in uncomplicated malaria was 4.81 IU/ml while in severe malaria 4.20IU/ml no significant difference was found (P value=0.083). No significant differences were detected between uncomplicated malaria and severe malaria categories. However, IFN-γ was found to correlate positively with Hb (P value=0.009), RBG (P value=0.001), temperature (P value=0.024), period of illness (P value=0.001) (correlations were analyzed by Pearson's correlation coefficient test), splenomegaly (P value=0.025) and with hepatomegaly
81 (P value=0.01) using Spearman' rank correlation coefficient test. Children with severe anaemia had a significantly low IFN-γ than those without severe anaemia (P value=0.000). Significant difference was found in IFN-γ between cerebral malaria and severe anaemia (P value=0.002) and between severe anaemia and repeated convulsions (P value=0.010) as well as between severe anaemia and mixed presentations (P value=0.032). About 14.9% of children were found to have IFN-γ values above 10 IU/ml of whom 46% present with repeated convulsions, 46% with UM and 8% with CM. Most of these patients were found to have low parasitemia.
Figure-15 IFN-γ in children with severe anaemia and those without severe anaemia
6
5
4
3
2
1
0 Mean IFN-gamma IU/ml SA w ithout SA
Severe anaemia
82 Figure-16 Correlation of IFN-γ with haemoglobin
83 Figure-17 Correlation of IFN-γ with random blood glucose
84 Figure-18 IFN-γ in different malaria categories
Table-26 Comparison of IFN-γ level in UM & SM categories UM SM categories Number Mean ±SD P value 4.81±9.43 Cerebral malaria 13 3.55±9.52 0.690 Severe anaemia 13 0.20±0.15 0. 087 Repeated 23 7.58±12.40 0.354 convulsions Hypoglycemia 1 0.50 0.656 Haemoglobinuria 2 4.85±6.43 0.995 Mixed 4 0.50±0.38 0..374 presentation
85 Table-27 Correlation of IFN-γ with clinical and laboratory parameters Parameter P value Age 0.967 Sex 0.976 Hepatomegaly 0.000 Splenomegaly 0.025 Period of illness 0.001 Temperature 0.024 Parasitemia 0.146 Hb 0.009 RBG 0.000 Insulin 0.527 C-peptide 0.752 TWBCs 0.103 Total bilirubin 0.302 Direct bilirubin 0.052 Indirect bilirubin 0.609 AST 0.408 ALT 0.363 ALP 0.277 Urea 0.124 Creatinine 0.475 Sodium 0.605 Potassium 0.495 IL-10 0.104 TNF-α 0.443 Outcome 0.754 Shaded boxes indicate significance IL-10 The mean of IL-10 in uncomplicated malaria was 237.71pg/ml while in severe malaria 201.67pg/ml no significant difference was found (P value=0.083). A Significant difference was found between children with severe anaemia and those with uncomplicated malaria. IL-10 was found to correlate with Hb level (P value=0.003) and negatively correlate with potassium (P value=0.044) when analyzed by Pearson's correlation coefficient test.
86 Figure-19 Correlation of IL-10 with haemoglobin
87 Figure-20 IL-10 in different malaria categories
Table-28 Comparison of IL-10 in UM & SM categories UM SM categories Number Mean ±SD P value 237.71±88.05 Cerebral malaria 13 213.80±91.53 0.421 Severe anaemia 13 171.73±95.51 0.032 Repeated 23 205.56±94.46 0.204 convulsions Hypoglycemia 1 286.70 0.588 Haemoglobinuria 2 147.95±193.53 0.197 Mixed 4 242.85±37.14 0.910 presentation Shaded box indicate significance
88 Table-29 Correlation of IL-10 with clinical and laboratory parameters Parameter P value Age 0.492 Sex 0.299 Hepatomegaly 0.728 Splenomegaly 0.270 Period of illness 0.051 Temperature 0.051 Parasitemia 0.072 Hb 0.016 RBG 0.224 Insulin 0.406 C-peptide 0.946 TWBCs 0.277 Total bilirubin 0.204 Direct bilirubin 0.467 Indirect bilirubin 0.211 AST 0.356 ALT 0.778 ALP 0.923 Urea 0.910 Creatinine 0.690 Sodium 0.860 Potassium 0.044 TNF-α 0.879 IFN-γ 0.104 Outcome 0.407 Shaded boxes indicate significance
Correlation of cytokines with age No significant correlation was found between age and the mean cytokines levels but when children were grouped into those below and above 5 years significant difference were detected in TNF-α and IFN-γ.
89 Figure-21 TNF-α in children below and above 5 years
400
300
200
100
0 Mean TNF-alpha(pg/ml) below 5 years above 5 years
age
Figure-22 IFN-γ in children below and above 5 years
5.5
4.4
3.3
2.2
1.1
0.0 Mean IFN-gamma(IU/ml) below 5 years above 5 years
age
90 Table-30 Cytokines in children below and above 5 years cytokine Below 5 years Above 5 years P value TNF-α 302.94±889.63 108.32±236.42 0.045 IFN-γ 3.54±8.59 5.20±10.24 0.034 IL-10 215.75±83.54 213.41±101.19 0.211 Shaded boxes indicate significance
91 CHAPTER FOUR DISCUSSION Introduction Severe malaria is a major health problem in Africa despite efforts directed toward its control. Children below 5 years are the main victim of severe malaria. The pathophysiological mechanisms underlying severe malaria manifestations are not well known. The aim of this study is to shed light on it so as to identify indicators of increased morbidity and mortality. The discussion will be mainly concerned with the serious complications of malaria recognised by WHO and the possible role and significance of cytokines in their pathogenesis. The effect of age on clinical categories of malaria and the cytokines response will also be taken in consideration. Effect of age on severity of malaria The mean age of children with severe malaria in this study was 56 months. The mean age of children with uncomplicated malaria was 82 months and a significant difference was found (P value=0.001). Children with severe malaria came from areas considered holloendemic for malaria. A study carried in Nigeria revealed 89.1% of children with severe malaria aged less than 5 years (242). Modlano et al (1998) conducted a study in Burkina Faso compared the age and clinical presentation of severe malaria between urban and rural areas, detected that children came from urban areas had a higher mean age than those came from rural areas (4.8 years versus 2.2 years). Children from rural areas had severe anaemia as the predominant manifestation while those from urban areas had CM (243).
In highly endemic areas (spleen rate in children from 2-9 years more than 50%) severe malaria tends to affect children younger than 5 years while
92 older children and adult tend to have the uncomplicated malaria (7). In low endemic areas (spleen rate in children 2-9 years less than 50%) severe malaria affects older children as well. Age is also relevant to the spectrum of severe malaria; severe anaemia and repeated convulsions are more prevalent in children below 5 years while cerebral malaria tends to affect older children (9, 149).
In this study children with severe anaemia and repeated convulsions were younger than those with cerebral malaria. This finding is in agreement with another study conducted by Reyburn et al (2005) in Tanzania aimed to describe the clinical manifestations and fatality of severe malaria at different transmission levels. It reported that age and level of exposure independently affect the clinical manifestation of severe malaria (244).
The differences caused by rate of transmission and age on the clinical picture of malaria can be explained by the time of acquisition of immunity. Immunity against malaria develops slowly with increasing age (245). As immunity developed, episodes of severe malaria decreased but still hyperparsitemia is frequent in older children. In adults parasitemia decreased and symptoms become few to absent.
Age has a prognostic value. In the study age was found to correlate negatively with outcome (P value=0.018). Children with increased morbidity and mortality were less than 5 years (39& 30months respectively). This is in agreement with other studies which detect young age as indicator of poor prognosis (243, 246).
93 Anaemia in severe malaria The parasite lives in red blood cells, replicates and at the end the schizonts rupture and destroy the RBC. Anaemia correlates with the degree of parasitemia and duration of the acute illness (155, 156). Immune system- mediated destruction also reported in malaria (158, 159, 160). Our findings support that hyperparasitemia was negatively correlates with Hb level and the duration of illness.
In the study, children with severe malaria had a significantly lower Hb level than those with UM (7.5 versus 9.9). Children with severe anaemia, mixed presentation and those with CM had significantly lower Hb in comparison with children having UM.
The pathogenesis of anaemia in severe malaria is multifactorial. RBCs destruction, suppression of erythropoeisis and cytokines are supposed to play a role in SA pathogenesis.
About 38.5% of children with severe anaemia were hyperparasitemic but only 2% of them had elevated bilirubin of indirect type which can be taken as indirect measure of RBCs destruction. This discrepancy can be explained by suppression of erythropoeisis by parasite- derived factors. Erythropoeisis suppression is apparent in the study by absent or inappropriate reticulocytosis in 15% of cases of SA. Erythropoeisis suppression by haemozoin was reported in malaria (173, 174). Haemozoin was found to inhibit erythroid development in vitro. In children with malarial anaemia, the proportion of circulating monocytes containing hemozoin is found to be associated with anaemia (173). Haemozoin mediates bone marrow suppression directly and through NO induction. Keller et al (2004) conducted a study in Gabonese children found peripheral mononuclear cells obtained from children with malaria
94 produce higher NO than healthy children and NO inversely correlated with Hb level (174). NO was found to suppress erythropoiesis (175) and to induce apoptosis in erythropoietic precursors (176).
Suppression of erythropoeisis by the cytokine TNF-α is another way of development of severe anaemia in malaria. TNF-α was found to suppress bone marrow function (180) and induce erythrophagocytosis (247). The suppressive effect of TNF-α on erythropoeisis may be mediated through NO. A study conducted by Maciejewski et al (1995) show that exposure of normal human bone marrow to NO result in inhibition of colony formation. NO induced apoptosis in bone marrow progenitors, as shown by electrophoretic detection of DNA degradation and deoxynucleotidyl transferase assay. Addition of IFN-γ or TNF-α increased inducible NOS mRNA and inducible NOS protein in bone marrow suggesting that NO may be one mediator of cytokine-induced hematopoietic suppression (248). In children with severe anaemia, TNF-α was found to be high (181). On the other hand, IL-10 may counteract the suppressive effect of TNF-α on the bone marrow by decreasing the production of TNF-α and stimulation of erythropoeisis (91). Children with severe anaemia had low IL-10(82, 182).
In This study children with severe malaria were found to have low IL-10 in comparison with UM. This is in accordance with other studies (82, 182). However, TNF-α was low in this group which did not agree with above mentioned studies but it is in line with another study that reported low level of TNF-α in children with severe anaemia (249).
IFN-γ values were found to correlate with Hb and the lowest values IFN- γ values were found in children with severe anaemia. IFN-γ prevents hepatocytes invasion by the parasites and thus decrease subsequent
95 development of erythrocytic stage (202, 203). It also stimulates the infected hepatocytes to produce NO which mediates intracellular parasite killing (250). Protection against anaemia may relate to decreased hemolysis and bone marrow suppression associated with episodes of clinical malaria. John et al (2004) assessed the IFN-γ response of peripheral blood mononuclear cells obtained from children residing in a holloendemic area in Kenya to LSA-1and its relationship to clinical episodes and the development of anaemia. The study revealed a significant association between IFN-γ responses and protection against clinical malaria and anaemia (251).
A study carried in Kenya proposed that IFN-γ production can be used as a potential marker of protective immunity against malaria associated anaemia in young children living in holloendemic areas (187). Our findings strongly confirm the finding of this study.
Severe anaemia was not associated with mortality and this may be related to blood transfusion which is the current treatment for severe anaemia. However, combination of severe anaemia with other malaria complications was found to increase morbidity. Another study detected increased mortality of severe anaemia when it was combined with respiratory distress or cerebral malaria (153).
Liver involvement in severe malaria
Liver pathology occurring during malaria infection is associated with the erythrocyte stage of the disease, although merozoites are not capable of infecting liver parenchymal cells. In contrast, infection of liver cells by sporozoites during the liver stage only leads to asymptomatic changes. This could be explained by the fact that during the erythrocyte stage, liver
96 and spleen are confronted with large amounts of infected erythrocytes and also free merozoites leading to a strong inflammatory response accompanied by the production of IL-12 and IFN-γ. Jacobs et al (2004) conducted a study on mice revealed that during infection T cells were accumulated in the liver parenchyma which was associated with an inflammation as indicated by elevated levels of liver enzymes (252). The pathology was found to be dependant on production of IL-12 and IFN-γ. CTLA-4 expression, a down regulator of T cells function, is induced on CD4+ T cells during the erythrocyte stage of malaria in humans as wells as in mice. Anti CTLA-4 antibody treatment leads to enhanced liver pathology and increased IFN-γ secretion. In addition kupffer cells and sinusoidal endothelial cells release IL-10 upon stimulation with lipopolysaccharides leading to down regulation of the proinflammatory response (253).
Hepatomegaly is common in children living in endemic areas. It is due to reticuloendothelial and lymphoid hyperplasia. However symptoms attributable to liver involvement in malaria were relatively uncommon (254) although elevation of liver enzymes was reported and malarial hepatopathy is now a recognized complication of malaria (255). It describes hepatic dysfunction in severe malaria and it is characterized by rise in serum bilirubin together with rise in serum transaminases 2-3 times above normal levels. The bilirubin is of the conjugated type and bilirubin level is usually more than 10mg/dl.
Bhave et al (2005) conducted a study in a tertiary hospital in Bombay and found an elevation of liver enzymes in 20% of patients with malaria (256). Anand et al (1992) detected jaundice in 39 patients and malarial hepatitis in 18 of them as indicated by a rise in their serum glutamate
97 pyruvate transaminase levels to more than three times the upper limit of normal and an absence of clinical or serological evidence to suggest drug or viral hepatitis. The liver in these patients was consistently enlarged. Liver histology showed Kupffer cell hyperplasia and deposition of malarial pigment. Plasmodium falciparum was demonstrated in sinusoidal red blood cells in only 2 cases (257). Another study revealed swollen hepatocytes, malarial pigment deposition, inflammatory infiltrates, congestion of hepatocyte and centrizonal necrosis along with elevation of transaminases in cases of malaria and jaundice (258).
In this study no significant differences were found between transaminase levels between UM and SM, however children with SM had a significantly higher frequency of high levels of these enzymes which were 1-2times above normal level. The conjugated bilirubin was not elevated so jaundice in the severe malaria children can not be attributed to liver dysfunction but most probably to haemolysis of the RBCs.
Further analysis of the data on liver functions and its relationship to cytokines was carried out and multiple regression models identified significant association between IFN-γ and ALP while TNF-α correlate positively with ALT. These findings suggest a possible role of these cytokines in the mediation of liver inflammatory process described by other studies. The mild liver inflammation in this group may also help explain the normal homeostatic regulation of glucose.
Bilirubin: In this study children with SM had significantly mild elevation of serum bilirubin than those with UM. It is mainly of unconjugated type. Children with increased morbidity and mortality had significantly higher serum bilirubin than those who had good outcome. Positive correlation was detected between bilirubin and urea and
98 creatinine (P value=0.000, 0.001 respectively) which may indicate contribution of hyperbilirubinemia to renal impairment within this group.
Hyperbilirubinemia of the unconjugated type usually result from haemolysis. Destruction of parasitized and non parasitized RBCs was known to occur in malaria. The parasite lives in red blood cells, use haemoglobin for their growth and replication and at the end schizonts rupture and destroy their erythrocyte host cells. Increased erythrophagocytosis was also reported in malaria and it is mediated by immunoglobulin and the complement system (158, 159, 160).
Presentation varies from mild jaundice to severe form with coma, renal failure and in some cases even hemorrhagic manifestations (259). It is only in this setting that jaundice signified a severe disease as noted by the World Health Organization action program. Marsh et al (1995) reported the presence of jaundice in children with malaria was one of serious prognostic factors (153). In this study, both serum bilirubin and liver enzymes indicate mild liver involvement
Spleen in severe malaria
The splenomegaly is usual in malaria and is often used as an epidemiological index of malaria transmission. Histologicaly both white and red pulp are expanded with reticuloendothelial and lymphoid hyperplasia, pigment deposition and sinusoidal dilatation (260). The spleen is considered the major site of parasite removal and splenic function is increased in acute malaria. Parasite clearance is delayed considerably in splenectomized patients (261). The spleen has a capacity to remove intraerythrocytic parasites leaving the host erythrocyte intact, a process known as biting which result in parasite clearance without
99 destruction of RBCs consequently prevent a decrease in Hb. Angus et al (1997)conducted a study in Thailand with the aim of detection of pf155 or ring erythrocyte surface antigen (RESA), which is expressed immediately following merozoite invasion as a measure of parasitization and correlated this with the presence of intraerythrocytic parasites. During acute falciparum malaria infection RBC containing abundant ring-infected erythrocyte surface antigen (Pf 155 or RESA), but no intracellular parasites, are present in the circulation. This indicates that in acute falciparum malaria there is active removal of intraerythrocytic parasites by a host mechanism in vivo (probably the spleen) without destruction of the parasitized RBC (154).
Chotivanich K et al (2002) found in acute malaria, RBCs that have been parasitized, but no longer contain a malaria parasite in the circulation. These are thought to arise by splenic removal of dead or damaged intraerythrocytic parasites and return of the intact RBCs to the circulation as 5 patients with acute falciparum malaria who had previously undergone splenectomy, circulating RESA-RBCs were absent in contrast to the uniform finding of RESA-RBCs in all patients with acute malaria and intact spleens. These observations confirm the central role of the spleen in the clearance of parasitized RBCs (262).
In this study splenomegaly occurred in 21.4% of children with severe malaria. Splenomegaly was found to correlate negatively with parasitemia and positively with Hb indicating that biting process may be the sole mechanism of parasite removal by spleen. IFN-γ correlated positively with splenomegaly and with haemoglobin indicating that IFN-γ may prevent the decrease in haemoglobin level through induction of
100 splenomegaly and hence enhance spleen parasite clearance capacity and reduce parasitemia.
TWBCs
High TWBCs counts can result in increased WBCs sequestration in the brain vessels which is one of the known theories of the pathology of CM. Depletion of alpha and beta lymphocytes protects against CM (102). Depletion of T regulatory cells in mice prevented the development of CM. The mice had reduced number of active CD4 and CD8 T cells in the spleen and lymph nodes with selective reduction of CD8 T cells in the brain suggesting T regulatory cells contribution to pathogenesis of CM by modulating the immune response (106).
In this study children with severe malaria had significantly higher TWBCs count than those with UM. CM, repeated convulsions and mixed presentation had significantly higher TWBCs count. Children with increased morbidity and mortality had high TWBCs count. This is in agreement with study carried by Maitland et al (2005) in Kenya found that children with fatal outcome had significantly higher WBCs count than survivors and they were frequently unconscious (213). Ladhani et al (2002) found leucocytosis was associated with both severity and mortality in children with falciparum malaria (263).
Monocytes adhered to the vascular endothelium was found to be associated with loss of astrocyte ensheathment of vessels and increase in BBB permeability that occurs at the time of neurological complications in the murine models of malaria suggesting that toxic products produced by monocytes might play a role in astrocyte degeneration (264).
101 Blood glucose homeostasis
The pathophysiology of malarial hypoglycemia in not well understood. Combinations of reduced endogenous glucose production and increased peripheral uptake of glucose were reported as possible causes (265). Unexplained impairment of gluconeogenesis in the presence of sufficient precursors was detected by a study on the payhophysiology of hypoglycemia in African children. The concentration of alanine, lactate and glycerol in children with hypoglycemia were not different from those without hypoglycemia (196). A study conducted in Khartoum Peadiatric Hospital (2009) detected high incidence of higher levels of gluconeogenic amino acids were associated with low RBG in hypoglycemic patients suggesting impaired gluconeogenesis (266). In contrast, Dekker et al (1997) conducted a study in children admitted to Kilifi District Hospital detected efficient gluconeogenesis which responded well to alanine infusion and proposed in severe malaria there is limitation in gluconeogenic precursors (200).
Although the RBG of children with SM in this study was within the normal physiological range (89.27mg/dl), it was significantly lower in this group than in children with UM. Hypoglycemia was an infrequent complication of falciparum malaria found in only 7% of cases of severe malaria. This is in agreement with another study conducted in Khartoum Hospital which revealed that hypoglycemia occurred in 4.4% of patients with severe malaria (266). A study carried in Gabonese children aimed to describe clinical and laboratory features of severe malaria found that hypoglycemia accounted for 6.2% of severe malaria cases (9). It also detected hypoglycemia, coma and hyperlactatemia as predictors of a poor outcome and the fatality rate increased in children with the three features.
102 In this study children with mixed presentation had lower blood glucose concentrations than children with UM as well as a prolonged course of the disease with increased hospital stay. On the other hand, children with hypoglycemia as the only presentation had short duration of illness and good outcome when managed by a protocol consisting of administration of 50% glucose and serial blood glucose monitoring.
Cytokines and hypoglycemia: Several workers linked cytokines to the pathogenesis of hypoglycemia. TNF-α was found to be high in children with hypoglycemia (191, 196, 200).
In this study children with hypoglycemia had the lowest TNF-α level which did not agree with the above studies. Moreover, TNF-α was found to positively correlate with RBG but correlation was not significant. The mean level of TNF-α in UM was 238.91pg/dl and RBG 108mg/dl. In severe malaria, the mean level of TNF-α was 178.52pg/ml and RBG 89.27mg/dl. TNF-α has been reported to be associated with decreased insulin secretion and increased peripheral resistance to insulin (25, 26); thereby it may well be a factor in the modulation of blood glucose in severe malaria. Mice with induced cerebral malaria with plasmodium berghei infection had significantly high blood glucose when TNF-α levels were high. Administration of recombinant TNF-α to normal mice caused an increase in blood glucose. Mice transgenic for human TNF-α did not develop hypoglycemia when infected with plasmodium yolli but showed signs of insulin resistance (196).
Feinstein et al (1993) examined the effects of TNF-α on the early events in insulin signaling pathway (267). He found that TNF decreased insulin- induced phosphorylation of both beta subunit of the insulin receptor and IRS-1, the major insulin cytosolic substrate. These changes were not
103 associated with any reduction in insulin binding. Moreover, TNF-α was found to inhibit the suppressive effect of insulin in hepatic glucose output in experiment conducted by Lang et al (1992) involved infusion of TNF-α in rats followed by infusion of insulin and glucose to maintain euglycemia. Glucose infusion rate necessary to maintain euglycemia was found to be 30% lower in TNF-α treated rats, indicating an insulin- resistant condition. This resulted from an impaired ability of insulin to both suppress hepatic glucose production and stimulate peripheral glucose uptake (25).
In this study IFN-γ was found to have a significant positive correlation with RBG and children with hypoglycemia had low level of IFN-γ. Negative correlation was found between period of illness and IFN-γ as well as with RBG indicating that early production of IFN-γ is necessary to counteract hypoglycemia. IFN-γ was found to decrease development of high density parasitemia and clinical malaria episodes (251). This may be caused by IFN-γ prevention of hepatocytes invasion and promotion of parasite killing within the hepatocytes. Schofield et al (1987) found that addition of IFN-γ to a hepatoma cell line invaded by malaria sporozoites lead to inhibition of parasite replication. The hepatoma cell line expressed IFN-γ receptors indicating that IFN-γ exerts its antimalarial activity by binding to surface receptors on hepatocytes and inducing intracellular changes unfavorable for parasite development (202). IFN-γ responses to liver stage antigen-1 (LSA-1) are thought to mediate this protection through induction of the nitric oxide pathway (268). In this study children with higher levels of IFN-γ were found to have low parasite densities. Positive correlation of IFN-γ was found with hepatomegaly and splenomegaly which enhance their parasite clearance capacity. IFN-γ was also found to have minor endocrine and metabolic
104 activities as it stimulate ACTH and cortisol secretion therby elevates blood glucose (201). It is apparent that IFN-γ may elevate the blood glucose through different mechanisms.
Parasitemia and hypoglycemia: Parasitemia was found to correlate negatively with RBG in this study. The frequency of hyperparasitemia was more in children with severe malaria than those with UM (30.4% versus 19.4%). Children with hypoglycemia and mixed presentation had higher frequency of hyperparasitemia than other severe malaria categories. It is well known that parasites use host blood glucose as a source of energy. Intraerythrocytic stages of malaria have a direct access to the plasma constituents through RBCs membrane pores and do not store glycogen or other reserve polysaccharides but depend on continuous supply of glucose from the host to continue the process of growth and replication (269). Suspensions of P .falciparum and P. vivax infected erythrocytes require the addition of glucose for parasite maintenance in vitro (270). Addition of glucose to P. vivax-infected cells increased the survival of the parasite (195). The plasmodia express a hexose transporter in their cell membranes. This transporter has a high affinity for glucose which maintains continuous supply of energy fuel to the parasites even when hypoglycemia complicates malaria (193). Parasitized erythrocytes consume up to 75times more sugar than their uninfected counterparts. The amount of glucose utilized is proportional to the degree of parasitemia (271).
Looking to the above findings and the suggested mechanisms of hypoglycemia by this study and others previous work, indicates that hypoglycemia in malaria results from different dependant and independent mechanisms acting together. Better understanding of these
105 mechanisms will allow development of drugs that can ameliorate hypoglycemia. Targeting parasite colonization mechanisms of the RBCs and parasite hexose transporter, may be a promising field for developing a new generation of drugs. Insulin and C-peptide Kawo et al (1990) detected that children with hypoglycemia had appropriately low insulin and C-peptide when compared to normoglycemic children (272). Dekker et al (1997) conducted a study in children with acute falciparum malaria admitted to Kilifi District Hospital (Keya) detected appropriately low insulin level in children with hypoglycemia (191). In this study no significant differences in mean insulin, C-peptide and I/C ratio were found between uncomplicated malaria and severe malaria. Children with hypoglycemia had insulin level in the lower limit of the physiological range.
Some studies, however, revealed that hyperinsulinemia is the cause of low blood glucose levels (195, 196). Elased et al (1994) carried a study in mice detected hyperinsulinemia at times of hyypoglcemia and dministration of diazoxide, insulin secretion inhibitor prevents development of hypoglycemia indicating the role of hyperinsulinemia in pathogenesis of hypoglycemia (195). Insulin secretion is stimulated directly by molecules derived from the parasite (195, 199) or it may complicate the treatment of children who receive quinine. Krishna et al (1994) detected that treatment with quinine was associated significantly with more episodes of post-admission hypoglycaemia when compared with artemether or chloroquine (188). Hyperinsulinemia was linked to quinine administration as detected by a study carried by White t al (1983) in eastern Thialand who detected inappropriate elevation of plasma insulin and C-peptide and a significant correlation between insulin and
106 quinine at the time of hypoglycemia (273). Quinine was found to stimulate insulin secretion, a finding came from a study carried by Agbenyega et al (2000) (198).
Our work confirms appropriate insulin response to hypoglycemia. However children with severe malaria had lower insulin value when compared to expected value indicating loss of the circulating insulin which may be result from immunologically mediated destruction or excessive loss through the kidneys which may result from the hyperdynamic circulation. Those with mixed presentation had significantly higher C-peptide level and lower I/C ratio than children with other malaria manifestations which may be explained by decreased insulin half life or decreased renal excretion of C-peptide within this group. Disturbances of electrolytes and renal functions Sodium: Hyponatremia was significantly more frequent in severe malaria (62.5%) compared to uncomplicated malaria (48.4%) A study carried on Kenyan children detected hyponatremia in 53% of children with SM. Hyponatremia was not related to peripheral parasite density, dehydration and abnormal renal function. ADH was found to be inappropriate to the degree of dehydration (217). Another study found increased urine sodium concentration and low osmolality in children with hyponatremia. The levels of ADH in these children were inappropriately high (218).
The present study reports hyponatremia, hypokalemia and low creatinine level in children with SM. These findings provide indirect evidence of inappropriate ADH secretion in severe malaria. No correlation was detected between the sodium level and degree of parasitemia.
107 Renal impairment was detected as contributor to hyponatremia in malaria (220, 274). Hyponatremia was found to be associated with increased urinary sodium concentration in the presence of reduced creatinine clearance in these patients (220). In this study no correlation was found between sodium and renal impairment so it is unlikely to be the cause of the hyponatremia reported above, however studies especially designed to investigate renal involvement may throw light on the mechanism of hyponatremia.
Potassium: Alteration in potassium is associated with acidaemia and alkalemia. A study carried in thirty-eight Kenyan children with severe malaria and acidosis detected that at admission, serum potassium was normal in 31 (81.6%), low in 4 (11%) children and 3 (6.3%) children had hyperkalaemia. Plasma potassium decreased rapidly after correction of acidosis. Fractional excretion of potassium and the transtubular gradient of potassium were above normal range, indicating renal potassium loss and study concluded that hypokalaemia is a common complication of severe malaria; however, it is often not apparent on admission but after correction of acidosis, plasma potassium decreases precipitously and thus serial monitoring of serum potassium is suggested in patients with severe malaria complicated by acidosis (216).
In the present study incidence of hypokalaemia is more frequent in children with SM than in those with UM. On the other hand hyperkalaemia was detected in 10.7% of children with SM and associated with increased risk of mortality. The death occurred in the first day of admission. It was more frequent in children with neurological manifestations. Changes in potassium levels in 56 Kenyan children (42 who survived and 14 who died) admitted to the hospital with clinical
108 features of severe malaria (impaired consciousness or deep breathing) complicated by acidosis, showed that hyperkalaemia complicated falciparum malaria in 9 children (16%), of whom 7 (78%) died, generally soon after admission (213).
In this study IL-10 was found to negatively correlate with potassium level. The stress of infection is known to activate the hypothalamic pituitary adrenal axis which results in increase cortisol secretion (275, 276). This is also reported in malaria (277, 279). On the other hand, the stress axis plays a significant role in regulating IL-10 expression. Inflammation of the central nervous system and endotoxaemia triggers the release of IL-10 in the circulation through activation of the stress axis (276, 279). This study states that activation of the stress axis during plasmodium infection may explain the relation between IL-10 and potassium observed in the study. Urea: Mild renal impairment is common in children with both complicated and uncomplicated malaria (280). Dehydration which was known to occur in malaria leads to acute renal tubular degeneration. Renal impairment is indicated by increased urea and creatinine concentrations (274, 280).
The present work shows that mean urea levels were nearly the same in SM and in UM. But children with mixed presentation had a significantly higher urea level than those with UM. This group of children had hyperbilirubinemia and a positive correlation was found between urea and creatinine with bilirubin indicating role of hyperbilirubinemia in the pathogenesis of renal impairment. This group of children had increased morbidity. Abdul manan et al (2006) found the presence of disseminated intravascular coagulopathy, cerebral malaria or hyperbilirubinemia in
109 patients with renal impairment were associated with poor prognosis (281). In the study children with mixed presentation in addition to the high urea and hyperbilirubinemia they had leukocytosis, low Hb and low RBG.
Acute renal failure is seen mostly in Plasmodium falciparum infection, but P vivax and P. malariae can occasionally contribute to renal impairment (280, 281). Malarial ARF is commonly encountered in non- immune adults and older children with falciparum malaria in areas of low endemicity. Several hypotheses including mechanical obstruction by infected erythrocytes, immune mediated glomerular and tubular pathology, fluid loss due to multiple mechanisms and alterations in the renal microcirculation have been proposed (209, 212, 207, 281).
Creatinine: In this study creatinine was found significantly lower in children with SM than in those with UM. This finding can be explained by the inappropriate ADH secretion which was reported to occur in SM and supported by the study.
Children with mixed presentation were found to have a relatively high creatinine in comparison with UM and other SM categories. This group of children had higher urea and relatively higher sodium in comparison with UM and other SM categories. They also had higher body temperature, higher incidence of tackycardia and tackypnoea than UM and other SM categories. These findings suggest that dehydration may play a role in this subgroup of SM and that dehydration is an indicator of poor outcome in children with SM. Thus volume resuscitation should be considered in severe malaria with evidence of dehydration. Maitland et al (2003) conducted a retrospective study in Kenya detected dehydration in 57% of children with SM. About 17.5% of these children died while only
110 7% of those without dehydration died (282). Maitland et al (2003) found that volume expansion in children with dehydration was associated with correction of the haemodynamic abnormalities and improvement of organ functions (283).
Haemoglobinuria
Two children in the study were found to have mild haemoglobinuria. They had no anaemia, jaundice or renal failure at admission but one of them developed jaundice in the ward. Massive haemoglobinuria is encountered rarely during the course of malaria. In this situation it can be accompanied by anaemia, acute renal failure and jaundice (226).
The haemoglobinuria in malaria is found to be related to quinine therapy. A study in African children report three cases of haemoglobinuria in children who repeatedly had malaria infection and quinine treatment (222). It is commonly encountered in Europeans living in Africa who take quinine irregularly (227). In this study one child had a history of an attack of malaria 2 weeks before admission and administration of quinine.
Parsitemia was found to be scanty or absent in haemoglobinuria (271). In the study parasitemia level was lower in haemoglobinuric patient than in other malaria categories.
111 Cytokines in severe malaria
The balance between pro- and antiinflammatory cytokines may be important in malaria presentation and outcome. Malaria tends to be more severe in children than in adults, because immunity develops with age. A number of cellular proinflammatory type 1 cytokines were significantly higher in all children (with or without malaria) than in all adults; these include the percentages of various lymphocyte populations making IL-6, both IL-6 and IFN-γ, or IL-8 (285). Therefore the immune response to malaria seems to be more proinflammatory in children than in adults. Low levels of IL-12 and TGF-β1 contribute to increased disease severity in malaria and IL-12 and TGF-β1 have been reported to be significantly lower in children with severe malaria. TNF α and IL-10 however, were significantly higher in children with severe malaria. In contrast, ratios of TGF-β1/TNF-α and IL-10/TNF-α were significantly lower in the severe malaria group (286). These data suggest that the inflammatory cascade in severe malaria is characterized by suppression of the protective effects of TGF- 1 and IL-12, and that overproduction of TNF-α may promote deleterious effects, such as severe anaemia (286) and cerebral malaria (287).The best-documented evidence implicates IFN-γ, TNF-α and IL-12 in the pathogenesis of CM (288) and a protective effect has been demonstrated for IL-10 (287)
A study on mice showed that the parasite induces expression of cytokines in the brain, liver and spleen which contribute to the pathological processes in these organs leading to differences in clinical features and lethal outcomes (289). Thèse cytokines are TNF α, IFN-γ, IL-1, IL-6, IL- 10 and IL-12. The higher levels are believed to be protective against infection. TNF-α and IFN-γ enhance clearance of parasitized cells by
112 phagocytosis (290). Intraperitoneal injection of mice with recombinant IL-12 appears to induce IFN-γ mediated protection against plasmodium yoellii sporozoite challenges (291). IL-10 inhibits production of TNF-α in response to malarial antigen, thus IL-10 appears to play an important role in counteracting the potentially harmful host proinflammatory response to malaria antigens (292). Alternatively cytokines may be involved in the manifestations of disease severity. Higher levels of IFN-γ, TNF-α, IL-1, IL-6, IL-10 and IL-18 are observed particularly in patients with severe malaria (293). TNF-α is known to be an essential element in the pathogenesis of cerebral malaria and severe malarial anemia (133, 91). Low IL-10 was found to be associated with severe anemia (82). The present study confirms the essential role of TNF-α in cerebral malaria as children with this complication had significantly higher TNF-α while children with severe anaemia had low IL-10.
Certain cytokines levels were found to predict the outcome. TNF-α level in serum can predict a fatal outcome in cases of CM. Patients with CM who had fatal outcome had TNF-α concentrations in serum higher than those with uncomplicated malaria (81). The ratio of anti-inflammatory (IL-10) to inflammatory (TNF-α) cytokines in serum is found to be the best determinant of the outcome (294)
In this study children aged less than 5 years in whom severe malaria predominate had higher levels of TNF-α when compared to those aged more than 5 years indicating essential role of TNF-α in pathogenesis of severe malaria. Children with cerebral malaria had a significantly higher TNF-α than other severe malaria categories. IL-10 was lower in children with severe malaria in comparison to those who had uncomplicated malaria although not statistically significant. However, IFN-γ was found to have a significant role in mediation of immunity against malaria as
113 children with uncomplicated malaria tend to have higher levels of IFN-γ when compared to those with severe malaria while children above 5 years had higher IFN-γ than children less than 5 years.
TNF-α TNF-α mediated immunity in malaria: TNF-α is produced by γδ T cells and NK cells in malaria during innate immunity. Early production of TNF-α is associated with protection (39). TNF- α has been shown to inhibit parasite survival in some rodent malaria models (295, 296). TNF- α and IFN-γ act synergistically to optimize nitric oxide production which in turn leads to parasite killing (53). In humans TNF-α and nitric oxide levels are associated with resolution of fever and parasite clearance (57, 58). TNF-α enhance human neutrophil killing of P. falciparum (295, 297, 298). TNF-α mediated pathology in malaria: Excessive production of TNF-α was found to be associated with increased risk of severe malaria. Lyke et al (2004) working in Mali found higher TNF-α level in children with severe malaria than children with UM (299). Another study carried in Senegal detected similar finding and higher level of TNF-α was associated with fatal outcome (300).
In this study the mean TNF-α level in children under 5 years was significantly higher when compared to those above 5 years. This finding is in accordance with the above literature that moderate levels of TNF-α are beneficial to the host and protect from severe disease while high level of TNF-α produce severe reaction which are damaging and predispose to severe malaria.
TNF-α in CM: Excessive TNF-α production plays a critical role in the pathogenesis of CM. This is supported by an experimental study
114 conducted by Grau et al (1992) revealed high serum TNF-α was at the time of the neurological syndrome and Single injections of anti-TNF-α antibody ameliorate the cerebral complications (133). Kwiatkowski et al (1990) undertook a study involving 178 Gambian children and found that plasma TNF-α level in children that had survived CM were twice as high as normal levels. In the fatal cases they were 10-fold higher than normal, leading to the conclusion that excessive TNF-α production may predispose humans to CM and its fatal outcome (81).
In situ hybridization shows an increase in expression of TNF-α mRNA in brain of mice with fatal cerebral malaria as early as day 3 post inoculation., which is several days before the onset of cerebral symptoms, although TNF-α protein was not detectable until at least day 5 post inoculation. At day 5 post inoculation small numbers of cells of microglia and astrocytes were positive for TNF-α protein. In addition, endothelial cells and monocytes adherent to the cerebral vasculature were also positive. At a time when the mice were showing severe cerebral symptoms at day 7 post inoculation there was a massive increase in the number of the above-mentioned cells positive for TNF-α protein (301).
In both human and murine CM, the blockage of cerebral vessels by parasitized RBCs is thought to be caused by TNF- α mediated up- regulation of adhesion molecules, such as intercellular adhesion molecule (ICAM)-1 on the endothelium (119, 131).
In this study children with cerebral malaria had a significantly higher mean TNF- α than those without cerebral malaria which agrees with the above literature. TNF-α in anaemia: Higher levels of TNF-α in serum were found to be associated with severe anaemia. Ortho et al (1999) found TNF-α and IL-
115 10 were significantly elevated in children with anaemia compared with children having UM and the highest concentrations of TNF-α were found in children with malaria anaemia. The mean ratio of IL-10 to TNF-α was significantly low in children with anaemia than in children UM (91). Thus, higher levels of IL-10 over TNF-α may prevent development of malaria anaemia by controlling the excessive inflammatory activities of TNF-α. Kurtazhals et al(1998) found that the ratio of TNF-α to IL-10 was significantly higher in fully conscious patients with severe anaemia than in children with CM and UM concluding severe malarial anaemia is a distinct disorder in which insufficient IL-10 response to high TNF-α concentrations may have a central role (82).
In this study children with severe anaemia had low TNF-α which doesn’t agree with the above literature. The explanation of this finding may be related to period of illness in severe anaemia group (4.46days) which allow TNF-α to exert its suppressive effect and then down regulated by IL-10. Another alternative explanation is the presence in this group of children of TNF-α allele associated with low TNF-α production. Low level of TNF-α in children with severe anaemia was reported in the presence of TNF-238A allele suggesting that genetic factors may influence TNF-α in SA (249). IL-10
IL-10 down regulates T helper 1 responses, namely IFN-γ and TNF- α secretion. A study in mice detected reduction in the amounts of lipopolysaccharide induced TNF-α release in the circulation after IL-10 pretreatment. IL-10 was given to mice before lipopolysaccharide challenge (302). Murine and in vitro studies have demonstrated IL-10 ability to inhibit TNF-α production in response to malarial antigens (303,
116 304). Thus, IL-10 appears to play an important role in counteracting the potentially harmful host proinflammatory response to malaria antigens.
IL-10 is thought to protect against severe malaria complications. IL-10 deficient mice infected with Plasmodium chabaudi suffer a more severe disease and exhibit a higher rate of mortality than control. The deficient mice showed a drop in body temperature to below 28°C and pronounced hypoglycemia. Elevated inflammatory responses have been shown to accompany pathology in infected IL-10 deficient mice. Neutralization of TNF-α in IL-10 deficient mice abolishes mortality and ameliorates the hypothermia, weight loss, and anemia but does not affect the degree of hypoglycemia. These data suggest that TNF-α is involved in some of the pathology associated with a Plasmodium chabaudi infection in IL-10 deficient mice (305). In this study children with severe malaria had lower IL-10 than children with uncomplicated but it did not reach significant statistical difference.
IL-10 in CM: IL-10 has a protective role against CM as it decreased TNF-α production. May et al (2000) conducted a study in Gabonese children found IL-10: TNF-α plasma level ratios <1 were a risk factor for both cerebral malaria and severe anaemia (294). During a Plasmodium chabaudi infection in IL-10 knockout mice, there is greater parasite sequestration, more severe cerebral edema, and a high frequency of cerebral hemorrhage compared with infection of C57BL/6 mice. Anti- TNF-α treatment ameliorated both cerebral edema and hemorrhages, suggesting that proinflammatory responses contributed to cerebral complications in infected IL-10 deficient mice (145). Furthermore kossodo et al (1997) found anti IL-10 anti body administration to resistant mice induced CM in 35.7% of these mice while administration of IL-10 to susceptible mice prevents the occurrence of CM (306).
117
One mechanism by which IL-10 prevents CM is through decreased platelet production. Platelets mediate a syndrome of cerebral malaria in an animal model of malaria infection (307). Histopathological studies of children who have died of severe malaria showed that platelet clumps with and without infected erythrocytes are frequently found in the vasculature (308). Infected erythrocytes may adhere to platelets, and the clumps of infected erythrocytes and platelets have been associated with severe disease (309). Platelets were found to accumulate in brain vessels of patients dying of CM and may play a role by providing adhesion receptors involved in sequestration (310). Casals –Pascaul et al (2006) studied the relationship between thrombocytopenia and cytokines in Kenyan children with severe malaria and showed that thrombocytopenia (platelet count < 150 x 109/L) strongly correlates with high levels of IL-10(311).
In this study the mean IL-10was lower in CM when compared to UM but it did not reach significance.
IL-10 in anaemia: Low level of this cytokine was linked to severe anaemia in children with malaria through inhibition of TNF-α production which was found to suppress erythropoeisis and mediate erythrophagocytosis.
Kurtzhals et al (1998) compared IL-10 and TNF-α between children with severe anaemia, cerebral malaria and uncomplicated malaria. Children with severe anaemia had significantly low level of IL-10 than other group and the ratio of TNF-α to IL-10 was high in severe anaemia than others concluding that severe malarial anaemia is a distinct disorder in which insufficient IL-10 response to high TNF-α concentration may have a
118 central role (82). In accordance with this study, a study carried by ortho et al (1999) who detected that the ratio of IL-10 to TNF-α was significantly higher in children with mild and high-density parasitemia than in children with malaria anaemia postulating that higher levels of IL-10 over TNF-α may prevent development of malaria anemia by controlling the excessive inflammatory activities of TNF-α (90)Nussenblatt et al (2001) found that in older children higher levels of TNF-α were significantly associated with higher IL-10 levels unlike younger children who had low IL-10. These data suggest that younger children do not maintain IL-10 production in response to the inflammatory process, and this mechanism may contribute to the more severe anemia found in younger children with malaria (312).
In this study severe anaemia group had a significantly lower IL-10 when compared to children with uncomplicated malaria and to those without severe anaemia. Furthermore it is correlated with Hb. These finding agrees with the mentioned studies.
In conclusion IL-10 is an important as anti-inflammatory cytokine in malaria counteracting the production and action of TNF-α in children with severe malaria. Genetic studies are highly recommended to explain the differences in the responses of children to the disease.
119 IFN-γ IFN-γ-mediated immunity in malaria: Most studies detect IFN-γ as the hallmark of immunity against malaria. Winkler et al (1999) investigated the type 1/type 2 dichotomy of T cells in patients of different age groups with acute P. falciparum malaria for the purpose of examining immunoregulatory mechanisms that ultimately result in different levels of antimalarial protection. He detected increased frequency of IFN-γ – producing cells in adult compared to children and in children aged more than 5 years in comparison to those aged less than 5 years (42). This finding was confirmed by the result of this study as the mean IFN-γ was significantly higher in children aged more than 5 years when compared to those aged less than 5 years. Furthermore IFN-γ production capacity of both CD8+ and CD4+ cells was positively correlated with aging Bucci et al (2000) conducted a study in children residing in Papua New Guinea compared IFN-γ response to three peptide epitopes of plasmodium falciparum liver stage antigen ( LSA) in different age groups. He detected that IFN-γ responses increased with age in malaria exposed subjects: 14– 16% and 30–36% of 2–5 and 6–54-year olds, respectively, had ≥10 IFN- γ-secreting cells/106 peripheral blood mononuclear cells when stimulated with at least one peptide variant of LSA. IFN-γ responses to all three peptides were also greater for older than younger individuals. No children <3 years old had lymphocytes that responded to all three peptides, whereas 45% of adults (mean age 48 years) had aggregated IFN-γ responses (314).
John et al (2004) postulate that IFN-γ responses to LSA-1 appear to require repeated P. falciparum exposure and/or increased age and are associated with protection against clinical malaria and anaemia (251).
120 IFN-γ is produced by γδ T cells and NK cells during innate immunity in malaria (38). Enhanced NK cell activity in spleens of mice infected with irradiated Plasmodium berghei sporozoites was detected (43). Plasmodium yoelii sporozoite infection has been shown to induce a rapid inflammatory response in the liver characterized by NK cell, macrophage, and T cell infiltration and IFN-γ production (44).
Early production of IFN-γ was associated with protection from severe disease. Mice deficient in γδ T cells had exacerbated early and chronic parasitemia (38). Another study detected a marked heterogeneity in magnitude of NK IFN-γ response reflecting variation between host capabilities to mount rapid proinflammatory response which then affect their innate suscepatability to malaria infection and its complication (40).
In this study as the incidence of high levels of IFN-γ was more frequent in uncomplicated malaria than in severe malaria. The mean IFN-γ in children above 5 years was significantly higher than those under 5 years.
IFN-γ plays an important role in resistance to blood stage malaria infection as well. Activation of human macrophages with IFN-γ results in activation of phagocytic activity and killing of malaria parasites (89). Peripheral blood mononuclear cells from children with uncomplicated P. falciparum infection produce high levels of IFN-γ when stimulated in vitro with merozoite antigens and these children have a lower risk of re- infection. In contrast, children with severe malaria show lower levels of IFN-γ production by Peripheral blood mononuclear cells and are more susceptible to re-infection (41). Mice defected in IFN-γ and its receptor show a predominantly Th2 response, which is associated with susceptibility to P. chabaudi infection (315, 316). Thus, IFN-γ is critical for resistance to blood stage malaria.
121 IFN-γ in severe anaemia: A study carried on Kenyan children with malaria examined IFN-γ responses to pre-erythrocytic stage antigens found that IFN-γ positive responders had higher haemoglobin levels and significantly reduced prevalence of severe malarial anaemia one month after the test compared with IFN-γ non-responders, suggesting that IFN-γ immune responses to these pre-erythrocytic antigens were associated with protection against malarial anaemia (187). The study proposed that IFN-γ production could be used as a potential marker of protective immunity against malaria associated anaemia in young children living in malaria holoendemic areas. IFN-γ induced the infected hepatocyte to produce NO which lead to killing of the intracellular parasite. This in turn leads to decrease in subsequent devoloping parasitemia. This study showed that IFN-γ correlate positively with haemoglobin. Children with severe anaemia had the lowest levels of IFN-γ. This indicates that high levels of the cytokine IFN-γ are needed to counteract anaemia.
IFN-γ in CM: IFN-γ has essential role in CM pathogenesis as it augments both the production and action of TNF-α. Grau et al (1989) conducted a study in mice detected that administration of monoclonal antibodies against IFN-γ after infection prevented the development of CM together with reduction in TNF-α production (142). Furthermore Rudin et al (2005) found that IFN-γ receptor deficiency in mice infected with Plasmodium berghei ANKA result in protection from CM. Plasmodium berghei ANKA -induced release of TNF-α and up-regulation of endothelial intercellular adhesion molecule expression, recruitment of mononuclear cells, and cerebral microvascular damage with vascular leakage occur only in wild-type mice not in mice with IFN-γ receptor deficiency. Resistance to CM in IFN-γ receptor deficient mice is
122 associated with reduced serum TNF-α level, reduced IL-12 expression in the brain and increased T-helper 2 cytokines (317).
In this study no significant difference was found in IFN-γ between children with cerebral malaria and those with uncomplicated malaria and in those without cerebral malaria. Genetic profile Genetic profile was found to affect cytokines responses in malaria. Ouma et al (2008) studied the relation between common variation in IL-10 promotor, circulating level of IL-10 and TNF-α and susceptibility to SA in Kenyan children. He detected that the -1,082G/-819C/-592C (GCC) haplotype was associated with protection against SA and increased IL-10 production. Although none of the other haplotypes were significantly associated with susceptibility to SA, individuals with the -1,082A/-819T/- 592A (ATA) haplotype had an increased risk of SA and reduced circulating IL-10 levels. The IL-10: TNF-α ratio was higher in the GCC group and lower in individuals with the ATA haplotype. The study concluded that common IL-10 promoter haplotypes condition susceptibility to SA and functional changes in circulating IL-10 and TNF- α level in children with falciparum malaria (313). May et al (2000) studied the association of allelic variants of the TNF promoter in children with severe malaria and IL-10: TNF-α ratio found that carriers of the wild type more frequently had an IL-10: TNF-α ratio >1. In contrast, individuals with a mutation at position 238 of the TNF-α promoter consistently had lower IL-10 than TNF-α plasma levels (IL-10: TNF-α ratio <1). These results show that, in children with severe malaria, TNF-α promoter variants influence the balance of IL-10: TNF-α in the plasma, which, in turn, affects the outcome in terms of clinical complications (294).
123 In this study significant difference was found in the mean TNF-α and IFN-γ between children with cerebral malaria and severe anaemia (P value=0.029 and 0.002 respectively) as well as in IFN-γ between severe anaemia and repeated convulsions (P value=0.010). Mcquire et al (1999) reported low level of TNF-α in children with severe anaemia while high levels were found in CM and TNF-238A allele was associated with SA suggesting that different genetic factors may influence CM and SA (249). Studies especially designed to link genetic profile with the different clinical presentation of malaria are recommended so as to identify children more likely to develop excessive inflammatory response and malaria complications. Genetic factors may also determine the receptor population for various cytokines, thus influencing the host response (85).
Cerebral malaria CM was found to be associated with increased morbidity and mortality. This finding is in agreement with another study conducted by Issifou et al (2007) detected CM as an indicator of poor prognosis (318). In 89.6% of cases coma is preceded by convulsions and they are associated with poor outcome. Molyneux et al (1989) examined the clinical and laboratory features of CM in 131 Malawian children detected convulsions at the time of admission, leucocytosis, hyperlactatemia and hypoglycemia to be associated with poor outcome (92). In this study children with CM had a significantly higher TWBCs count, higher bilirubin and lower Hb in comparison with those with UM.
CM is encountered in older children who probably had acquired significant degree of immunity against malaria if they were residing in endemic areas (32). The mean age of children reported in this study with CM was 61 months. This suggests that CM is an immune system related
124 disease. Over production of cytokines TNF- α and IFN-γ causes many of the pathological features of CM. Sequestration of the parasitized RBCs and leukocytes in the cerebral microvasculature was reported as a cause of CM (110). A study carried in mice detected sequestration of parasitized RBCs and leukocytes in brain vessels was associated with increased expression of TNF-α and ICAM-1 in blood vessels (111). TNF- α upregulates adhesion molecules involved in parasitized RBCs and leukocytes sequestration within the cerebral microvasculature (119). High TNF-α in the serum was detected by many studies of CM (130, 133).
Indirect evidence for parasite sequestration in this study can be deduced from comparing hyperparasitemia and bilirubin in CM and SA
Severe malaria Hyperparasitemia(percentage) Bilirubin(mean in category mg/dl) CM (n=13) 61.5 1.162 SA (n=13) 61.5 0.892
The difference noted may be explained by the observation that the peripheral parasite count in CM is not a real indicator of parasitemia as the parasites are sequestered in brain. This finding is in accordance with another study carried by Seydel et al (2006) who measured the concentration of parasite-derived lactate dehydrogenase (pLDH) in tissue samples obtained at autopsy from patients with clinically defined cerebral malaria. There was no correlation between peripheral parasitemia and the measurement of pLDH in any of the tissue samples (319).
Another mechanism by which TNF-α is supposed to cause CM is through reduction of cerebral blood flow. Reduced cerebral blood flow was detected by MRI at advanced stages of CM and result in neuronal damage as apparent from reduction in level of N-acetylaspartate (98, 320).
125
MRI study revealed that focal intrastriatal injection of TNF-α in mice leads to significant decreases in cerebral blood volume (320). Moreover, TNF-α facilitates the release of endothelin by endothelial cells (321). Endothelin is a potent vasoconstrictor and has a capability to increase blood brain barrier permeability which leads to cerebral edema and by the two mechanisms endothelin reduces cerebral blood flow (116, 322, 323). Excessive endothelin expression was found in brain of infected mice (103). Microglial cells activation is also reported in malaria. Studies carried in mice detected morphological changes in microglia consistent with their activation and redistribution toward blood vessels (140). The activated microglia through an excitotoxic mechanism have been suggested to cause the pathological and clinical feature of CM (141). Microglial cells activation was found to be induced by TNF- α (139).
TNF- α was found to induce NO production (134). Increased expression of inducible nitric oxide synthase was found during acute CM in the neurons, microglial cells, astrocytes and in the macrophages surrounding ring hemorrhages (136). NO metabolites were found to be higher in plasma and the CSF of children with CM and associated with poor outcome (137, 65, 138). NO is known to affect synaptic transmission (135) and thus it may contributes to the developing seizures and coma.
So TNF- α through different mechanisms which can act alone or in combination in a given individual may have a significant contribution to the pathological and clinical features of CM.
High level of IL-10 is thought to be protective against CM development by inhibiting TNF-α production. IL-10 knockout mice infected with
126 plasmodium chabaudi develop greater parasitic sequestration and severe cerebral edema and hemorrhages and administration of anti-TNF ameliorate both cerebral oedema and haemorrhages (145).
In this study significant difference was found in the mean TNF-α between children with cerebral malaria and those without cerebral malaria. Furthermore children with cerebral malaria had lower ratio of IL-10 to TNF- α when compared to those with uncomplicated malaria (0.99 versus 0.82). This indicates that children with CM had lower IL-10 to balance the inflammatory effect of TNF- α.
IFN-γ was found to augment the production and action of TNF-α. An experimental study in mice detected that administration of monoclonal antibodies against IFN-γ after infection prevented the development of CM and its associated histological feature. Monoclonal antibodies administration also resulted in decreased TNF-α production which leads to the conclusion that IFN-γ both augments the production and action of TNF-α (142). In this study although mean IFN-γ is higher in CM when compared to UM and other categories of SM, it was not statistically significant.
Repeated convulsions Repeated convulsions represented 41% of cases of SM which was the most frequent complication of SM in this cohort. Children with this complication had a mean age of 56 months. A retrospective study conducted by Idro et al (2007) in Kilifi Hospital in Kenya reported seizures to be the most common neurological feature of acute falciparum malaria and were observed at admission in 37.5% of children. Seizures were not common before the age of 6 months. After 12 months, the age-
127 specific prevalence increased rapidly reaching a peak prevalence of 48.6% among patients aged 27 to 33 months (324).
Repeated convulsions in malaria were found to indicate cerebral dysfunction rather than being febrile convulsions as no correlation was detected between fever and the incidence of repeated convulsions (150). Similar findings are reported in this study.
Prolonged, multiple seizures complicate a high proportion of cases of childhood cerebral malaria lead to further deterioration in the level of consciousness. Repeated convulsions were found to contribute to both the morbidity and mortality (93). In this study children with increased morbidity (three children with mixed presentation and one child with CM) and mortality (one child with CM) had repeated convulsions. However children with isolated repeated convulsions had a more favorable outcome.
In the present study children with repeated convulsions were found to have significantly higher bilirubin and higher total WBCs count in comparison with UM. They had TNF-α level comparable to UM but relatively higher levels of IFN-γ. The conclusion that can be derived from these findings is that the mechanisms of CM may be the same mechanism of repeated convulsions. Period of illness before admission may discriminate the two. The mean period of illness in children with repeated convulsions was 1.61 days while in CM it was 2.23 days. Early presentation to hospital allows early antiparasitic therapy which is known to decrease cytokines level (199). Another factor may be the low parasite density which was observed in children having repeated convulsions in comparison to those with CM (17.4 versus 38.5). It appears that multiple factors are involved in the pathogenesis of repeated convulsions. This
128 indicates that future research should focus on single presentation so as to determine the pathophysiological mechanisms.
Indicators of poor outcome Malaria is one of the major killers of children in Africa. About 94% of global deaths attributed to malaria occur in Africa (325). Despite the programmes of malaria control the incidence of mortality continues to rise (326). This is mainly attributable to the fact that the pathophysiological mechanisms of the disease is still not well known despite the huge literature which has not yet become reflected in the management protocols.
Many studies tried to find indicators of severity of disease and markers for morbidity and mortality. The ultimate aim is to identify children with increased risk of higher morbidity and mortality and thereby offer these children intensive care taking into account the morbidity and mortality indicators.
Marsh et al (1995) et al conducted a prospective study in the pediatric ward of a district hospital in Kenya detected mortality rate of malaria was 3.5% and reported that 84% of the deaths occurred within 24 hours of admission. Key prognostic indicators were impaired consciousness, respiratory distress, jaundice and hypoglycemia (153).
Dzeing- Ella et al (2005) examined the clinical and laboratory features of severe malaria in Gabonese children and found the prognostic indicators with the highest case fatality rates were coma/seizures, hyperlactataemia and hypoglycaemia and the highest case fatality rate was in children with all three of these features (9).
129 Another study in Tanzania detect hypoglycemia, respiratory distress and dehydration as indicators of increase risk of death (327)
In this study multiple regression models identified severe malaria, cerebral malaria, mixed presentation and potassium as independent factors associated with increased morbidity and mortality. The mortality rate was 1.8% and death occurs within the first day of admission.
Cerebral malaria: CM was found to predict both fatal outcome and morbidity. This is in accordance with another study conducted by Winkleret al (2008) in children which reported CM and respiratory distress independently associated with increased risk of morbidity and mortality (328).
In this study hyperkalemia was found more frequent in children with neurological involvement than in other malaria categories. Furthermore it is associated with increase risk of death within this group.
Maitland K (2005) et al detected that at the time of admission to the hospital, hyperkalemia may complicate cases of acidosis due to severe malaria and is associated with high, early mortality. Patients with fatal cases were significantly more frequently unconscious (213).
It is also relevant to note that children with CM had significantly lower Hb level and higher bilirubin level and TWBCs count than those with UM.
Mixed presentation: Four children in the study were found to have more than one WHO criteria of severe malaria. The table below shows the clinical features of this group.
130 Case No CM Repeated Severe Hypoglycemia convulsions anaemia 35 absent present present present 39 absent present absent present 73 present present present absent 82 present present absent present
This group had significantly lower RBG, Hb and higher bilirubin, total WBCs count and urea compared to children with UM.
Idro et al (2007) from study in Kenya found that children having hypoglycemia, acidosis, impaired perfusion, hyperparasitemia in association with neurological involvement increased mortality and neurological sequelae (324).
Maitland et al (2005) reported that children with higher median shock score, higher WBC count and worse metabolic derangements (acidosis, hypoglycemia, hyperkalaemia, and increased creatinine levels) had higher risk of death than those with lower levels of these parameters (213).
Children having disseminated intravascular coagulopathy, cerebral malaria or hyperbilirubinemia in addition to renal impairment were found to have poor prognosis (281). Children with mixed presentation in this cohort had higher urea and creatinine in comparison to those with UM indicating renal impairment and poor prognosis.
Potassium: In the study hyperkalaemia occured in 10.7% of SM cases and it is associated with increased mortality. A study in Kenyan children found that hyperkalaemia complicated falciparum malaria in 16% of whom 78% died soon after admission (213).
131 Table-34 Indicators of poor prognosis reported by some studies Parameter Ella- Schellenb Marsh Winkle Maitlan Abdulma Dzein erg et et ret al d et al nnan et al g et al(1999) al(199 (2008) (2005) (2006) al(200 5) 5) CM Yes - Yes Yes - Yes Repeated Yes - - - - - convulsion s Hypoglyce yes Yes - - Yes - mia SA ------Respiratory - yes yes Yes - - distress Hyperkalae - - - - Yes - mia Sodium ------Urea - - - - - Yes Creatinine - - - - yes yes
In conclusion, severe malaria is a multisystem disorder even when the most dramatic disorder appears to involve a single organ. Many pathophysiological mechanisms may act at the same time and at the same individual to result into the clinical category of severe malaria. Dysregulation of the immune system may have a critical role in determination of clinical category of malaria which was acquired by child. This shed light on genetic background which may affect the clinical presentation of the child.
132 CONCLUSIONS Age distribution 1-Severe malaria is more common among children less than 5 years of age. 2-Age correlates negatively with the outcome: more morbidity and mortality was found among the younger age group. 3- Severe anaemia and repeated convulsions occurred in the younger age group. 4- Cerebral malaria occurred in the older age group. 5-Haemoglobinuria occurs in the older age group Blood glucose 1- Children with severe malaria had significantly lower RBG than those with UM. However it was still within the normal physiological range. 2- Hypoglycemia was an infrequent complication which represented 7% of severe malaria cases. 3- Higher parasitemia levels were significantly correlated with low blood glucose which may be due to consumption of the glucose by the parasites. 4- Hypoglycemia alone was not associated with morbidity and mortality but when combined with other malaria complication it leads to increased risk of morbidity and mortality. 5- IFN-γ correlates positively with blood glucose which suggest a role in counteracting hypoglycemia. This is a possibility due to its effect on release of ACTH and cortisol. IFN-γ was also found to induce spleen and liver enlargement which enhance their clearance of the parasites from the blood stream. Insulin & C-peptide 1-No significant differences were found in the mean insulin and C- peptide between children with uncomplicated and severe malaria.
133 2- Although ratio of I/C was lower in children with severe malaria, no significant difference was found when compared to the ratio of children with uncomplicated malaria. 3- The expected value of insulin was much higher than the recorded value indicating a decreased half life of insulin in severe malaria. 4- Insulin was positively correlated with RBG which indicates an appropriate response. 5- Children with mixed presentation had a significantly higher C-peptide and lower I/C in comparison to those with uncomplicated malaria and those with single presentation which may be due to failure of renal excretion of C-peptide in this group of children. Haemoglobin 1-Children with higher parasitemia level had significantly low Hb indicating direct hamolysis of parasitized RBCs as a cause of anaemia. 2- IFN-γ and IL-10 correlate positively with Hb indicating the essential role of both cytokines in counteracting anaemia. 3- Positive correlation was found between Hb and splenomgaly and hepatomegaly. This can be explained by removal of the parasites from infected RBCs without destroying them (biting function of the spleen). This result suggests that biting can occur in the liver also. Liver functions 1- No significant difference in serum transaminases was found between UM and SM. 2- Children with SM had a higher incidence of elevated transaminases indicating liver dysfunction. 3- High levels of TNF-α and IFN-γ were found to associate with high serum transaminases l indicating involvement of these cytokines in mediation of liver pathology.
134 4-Children with severe malaria had unconjugated hyperbilirubinemia indicating haemolysis as the cause. Increased morbidity and mortality was associated with high levels of bilirubin. TWBCs 1-Children with severe malaria had higher TWBCs count than those with UM. 2- Children with increased morbidity and mortality had higher TWBCs count. 3- Children with CM, repeated convulsions and mixed presentations had a significantly higher TWBCs count in comparison to those with UM indicating essential role of white blood cells in the pathogenesis of severe malaria through sequestration in the blood vessels or through secretions of excessive proinflammatory cytokines or toxins. Cytokines 1. IFN-γ: IFN-γ was a hallmark of immunity against malaria as indicated by the following. • Children above 5 years had higher IFN-γ level than those less than 5 years indicating that children produced more IFN-γ were protected against severe malaria. • Low levels of IFN-γ were associated with presentation of severe malaria. • Higher levels of IFN-γ were associated significantly with higher RBG and Hb. Children with severe anaemia had a significantly lower IFN-γ than those without severe anaemia. So IFN-γ protects children from two severe malaria categories; hypoglycemia and severe anaemia and this effect was most pronounced in early producers of IFN-γ. • IFN-γ correlates positively with splenomegaly and hepatomegaly which may improve their clearance of the parasite.
135 • Children with severe anaemia produced significantly lower IFN-γ than children with cerebral malaria and repeated convulsions. 2. TNF-α • High level of TNF-α was associated with the risk of severe disease. Children below 5 years had significantly higher level than those above 5 years. • Children with CM had a significantly higher level of TNF-α than those without CM indicating essential role of this cytokine in pathogenesis of CM. • Children with severe anaemia produced significantly lower TNF-α than those with cerebral malaria. 3. IL-10 • IL-10 is essential in prevention of SA development in children with malaria as IL-10 level was found to be low in SA and IL-10 correlate positively with Hb. • IL-10 correlate negatively with potassium. Activation of the stress axis may explain this finding. Renal functions Urea 1- No significant difference in urea level was found between severe and uncomplicated malaria. 2- Children with mixed presentation had a significantly higher urea level. In the presence of relatively high creatinine and sodium within this group, renal impairment may be explained by dehydration resulting in renal hypoperfusion. 3- Children with increased morbidity had higher urea level than those with UM which indicates that renal impairment is an favorable prognostic factor.
136 Creatinine 1-Children with severe malaria had lower creatinine level than those with UM suggests increased renal clearance. 2-In the presence of hyponatremia and hypokalemia, low creatinine can be attributed to the syndrome of inappropriate ADH secretion, hyperdynamic circulation or both. Sodium 1-Incidence of hyponatremia was more frequent in SM than in UM. This may indicate inappropriate ADH secretion in severe malaria. Potassium 1-Incidence of hypokalaemia is more frequent in SM than in UM which is in favor of inappropriate ADH secretion. 2-Hyperkalaemia was found in 10.7% of severe malaria cases. 3-Most of children with hyperperkalaemia had neurological manifestation and increased mortality Indicators of morbidity and mortality 1-cerebral malaria, mixed presentation, young age and high level of potassium were independently associated with increased risk of morbidity and mortality. 2-Laboratory parameters significantly associated with cerebral malaria and mixed presentation were high bilirubin, TWBCs count, high urea, high creatinine and low Hb and low RBG.
137 RECOMMENDATIONS
• Children with severe malaria should be admitted to an ICU facility where protocols of management of severe malaria are clearly defined. • Children with severe malaria aged below 5 years are at higher risk and should be offered special care. • Children with cerebral malaria and mixed presentation need to be monitored more intensively. • Potassium and sodium instability need to be monitored on admission and during the first 24 hours as frequently as possible. • RBG, Hb, urea, bilirubin estimation and TWBCs count should be done on admission for any child with severe malaria. • Studies linking the clinical presentation with genetic profile are recommended especially those related to cytokines receptors. • Studies of renal involvement in severe malaria are highly recommended. • A pilot study should be considered for children with severe anaemia to recieve treatment with a combination of anti-parastic therapy, IFN-γ and IL-10 administration • A panel of experts is recommended to be set up by the Ministry of Health Malaria Program and the Sudan Association of Peadiatricians so as to revise the present protocol for management of severe malaria in the light of recent evidence from Sudanese and African studies. • Animal model are needed for more focused studies of the effects of cytokines.
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187 APPENDIX University of khartoum Faculty of Medicine Department of Physiology Pathophsiology of severe malaria in children: clinical and laboratory correlations Questionnaire
Name: ……………………………………………………………………. Age: ……………………………………………………………………. Sex: ……………………………………………………………………… Residence: ………………………………………………………………... Informant: ……………………………………………………………... c/o: ………………………………………………………………………... History of present illness:
Systemic review: cardiopulmonary system: Cough: S.O.B: Gastrointestinal tract: Nausea: Vomiting Diarrhea: Abdominal pain central nervous system: Loss of consciousness Duration>30 min <30 min Convulsions: Single: Repeated: Urinary system Frequency of Micturition Colour of Urine Volume of Urine Abnormal bleeding sites
188 Past medical history: Previous attacks of malaria Diabetes mellitus Any chronic illness physical examination Pallor Jaundice Cyanosis Vital Signs: PR RR Temperature Wt Chest: Movement: Equal Less in Rt Less in Lt Expansion: Equal Less in Rt Less in Lt Trachea: Central Deviated to Rt Deviated to Lt Tactile focal fremitus------Breath sound: Vesicular Bronchial Added sounds: Crepitating Rhonchi cardiovascular system: apex beat Heart sound Added sound Heart failure Abdomen: Distension Renal angles mass Tenderness----- Ascites Signs of hepatic failure: Foetur hepaticus Flapping tremor Liver span CNS: Level of consciousness Cranial nerves Motor and sensory systems Coordination
189 Investigation: BFFM Species Parasitemia Hb TWBCs RBS
Urine examination: Protein Sugar Acetone Hb Renal function test: S.creatinine S.urea S. NA+ S.K+ Liver function test: AST ALT Bilirubin Cytokines: IL-1 IL-6 IL-10 IL-12 TFN- α Follow-up - General condition - PR - RR - Temp
- Outcome Survived Discharged in day Died in 1st day 2nd day on day
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