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The Prevalence of Vector Borne Diseases in Tampin Hospital , Negeri Sembilan

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The Prevalence of Vector Borne Diseases in Tampin Hospital , Negeri Sembilan THE PREVALENCE OF VECTOR BORNE DISEASES IN TAMPIN HOSPITAL , NEGERI SEMBILAN GANGA DEVI A/P B. SINNIAH UNIVERSITI SAINS MALAYSIA 2018 THE PREVALENCE OF VECTOR BORNE DISEASES IN TAMPIN HOSPITAL , NEGERI SEMBILAN by GANGA DEVI A/P B. SINNIAH Thesis submitted in fulfilment of the requirements for the degree of Master of Sciences July 2018 ACKNOWLEDGEMENT This author is very grateful to GOD ALMIGHTY for without His grace and blessings, this study would not have been possible. My deepest appreciation to my supervisor, Associated Professor Dr. Zary Shariman Yahya, for his excellent guidance, his critical insights, motivation and his patience, willingness of time in sharing knowledge. He was extremely encouraging and instrumental in developing various ideas and arguments of this thesis. Thanks a lot Dr Zary Shariman, for giving me chances as a part time Master Student under your supervision. I sincerely thank the Director of Tampin Hospital, Dr Hajjah Hatijah Bt Hj Mohd Tan for her guidance and encouragement in carrying out this project work. I also wish to express my gratitude to the staffs at Tampin Hospital who have rendered their help during the period of my study. I would like to convey my gratefulness to all my friends, Rajiv and Azirah for their support and help during my experiment in laboratory. The skills and knowledge which I have gained throughout my experiment I perceive as very valuable component in my future development. Special thanks to the practical students in Lab 418 for inspiring and encouraging me as I was an older student among them. Finally, I would like to acknowledge with gratitude, the support and love of my husband and children, my mother, brothers and also sisters. They all kept me going, and this thesis would not have been possible without them. ii TABLE OF CONTENTS ACKNOWLEDGEMENT………………………………………….…... ii TABLE OF CONTENTS……………..………………………………… iii LIST OF TABLES.……………………………………………………... iv LIST FIGURES.………………………………………………………… v LIST OF DIAGRAM………………………………………………….... xv LIST OF ABBREVIATIONS.………………………………………….. xvi ABSTRAK.……………………………………………………………... xviii ABSTRACT.…………………………………………………………..... xx CHAPTER 1-INTRODUCTION 1.1 Problems Statement…...……………………………………………... 3 1.2 Research Objectives………………………………………………….. 5 1.2.1 General Objective……………………………………………... 5 1.2.2 Specific Objective…………………………………………….. 5 CHAPTERS 2-LITERATURE REVIEW 2.1Vector Borne Disease………………………………………………... 6 2.1.1 Introduction………………………………………………….. 6 2.1.2Transmission of Vector-borne diseases in Malaysia…………... 9 2.2 Major prevalent vector-borne diseases in Malaysia………………… 25 2.2.1 Malaria………………………………………………………… 28 2.2.1(a) The Transmission of Malaria Cycle…………………... 30 iii 2.2.1(b) Type of Malaria Parasites…………………………….. 32 2.1.1(b)(i) Plasmodium falciparum ………..………… 33 2.1.1(b)(ii) Plasmodium vivax…………………….….. 35 2.1.1(b)(iii)Plasmodium malariae .…….…………….. 37 2.1.1(b)(iv)Plasmodium ovale…………..……………. 38 2.1.1(b)(v)Plasmodium knowlesi…………..…………. 39 2.2.1(c) The Morphology of Malaria’s Plasmodium spp……… 40 2.1.1(d) Clinical Features……………………………………… 41 2.1.1(e) Laboratory Finding……………………………………. 41 2.2.2 Dengue………………………………………………………….… 43 2.2.2(a) Dengue Viral Transmission Cycle……………………..... 45 2.2.2(b) Dengue Viral…………………………….……….……… 47 2.2.2(c) Clinical Features………………………….……………... 52 2.2.2(c)(i) Dengue Fever (DF)………………….…..…... 54 2.2.2(c)(ii) Dengue Hemorrhagic Fever (DHF)….…….... 55 2.2.2(c)(iii)Dengue Shock Syndrome (DSS)….…….…... 56 2.2.3 Filariasis………………………………………………………….. 57 2.2.3(a) Type of Filaria worms …………………………….……. 61 2.2.3(a)(i )Wuchereria bancrofti…………..……………... 62 2.2.3(a)(ii)Brugia malayi………………….……….……. 64 2.2.3(a)(iii)Brugia timori………………………….….…. 65 2.2.3(a)(iv)Brugia pahangi……………….……………... 68 2.2.3(b) The Transmission of Filariasis ………………………….. 70 2.2.3(c) Clinical features…………………………………………. 72 2.2.3(d) Laboratory Finding……………………………………… 72 iv CHAPTERS 3-METHODOLOGY 3.1 Blood Sampling………………………………..……………………... 73 3.2 Subject and Inclusion Criteria……………………………………….. 75 3.3 Laboratory Procedures………………………………………………. 75 3.3.1 Blood Samples…………………………………………………. 75 3.3.2 Full Blood Count (FBC) method………………………………. 76 3.3.3 Patient Diagnosis………………………………………………. 76 3.3.4 Dengue Serology Screening method…………………………… 77 3.3.5 Blood Film Malaria/Filaria Parasite Examination……………... 79 3.3.6 The molecular analysis of Dengue Viruses and Malaria parasites in Blood Samples ……………………………………. 80 3.3.6(a) RNA extraction of dengue virus in blood samples……. 80 3.3.6(a)(i) Agarose gel electrophoresis……………….. 80 3.3.6(a)(ii) Polymerase Chain Reaction (PCR)……….. 83 3.3.6(a)(iii) Polymerase Chain Reaction (PCR) after Optimization……………………………… 8 7 3.3.6(b) DNA extraction of parasites for confirmation after Blood film examination…………………………….…. 90 3.3.6(b)(i)Agarose gel electrophoresis………………... 91 3.3.6(b)(ii)Polymerase Chain Reaction (PCR) ……….. 91 3.3.6(b)(iii)Polymerase Chain Reaction (PCR) after Optimization ………………………………………. 95 3.3.7 Gel extraction of PCR products……………………………….. 98 v 3.3.8 Optical Density (OD) Determination………………………….. 99 3.3.9 Sequencing……………………………………………………... 100 3.3.10BLAST………………………………………………………… 101 3.3.11Statistical Analysis……………………………………………. 102 3.3.12Socio-economic and Environmental Factors …………………. 103 CHAPTERS 4-RESULTS 4.1 The Prevalence of Vector Borne Disease Cases In Tampin Hospital... 104 4.2 Dengue serology and PCR screening for Dengue infection in blood samples ………………………………………………………………. 107 4.2.1 Major Ethnic groups involved in blood samples……………….. 108 4.2.2 Dengue Serology Test………………………………………….. 109 4.2.3 Molecular Identification and confirmation of Dengue Virus ….. 103 4.2.4 Confirmation of Dengue Virus Serotype ………………………. 114 4.3 Malaria Microscopic Examination and PCR test…………………….. 115 4.3.1 Microscopic Examination of Malaria…………………………... 116 4.3.2 Molecular Identification and confirmation of Malaria parasite… 117 4.4 Filariasis Microscopic Examination and PCR test…………………… 120 4.5 The Clinical Symptoms in Patients…………………………………... 120 4.6 Laboratory blood analysis……………………………………………. 122 4.7 Socio-economic and Enviromental Factors ………………………….. 124 CHAPTERS 5-DISCUSSIONS 5.1 Socio-economic and Enviromental Factors…………………………... 131 vi 5.2 Full Blood Count (FBC)……………………………………………… 139 5.3 Dengue Serology Test………………………………………………... 143 5.4 Blood Film Microscopic Examination of Malaria (BFMP)………….. 148 5.5 Molecular Identification of Malaria and Dengue…………………….. 152 5.5.1 DNA identification of Malaria, Plasmodium knowlesi………… 152 5.5.2 RNA identification of Dengue Virus, DENV 4………………… 154 CHAPTERS 6-CONCLUSION 6.1 Conclusions…….........………………………………………………... 157 6.2 Recommendations for further research……………………………….. 159 REFERENCES………………………………………………………...... 161 APPENDICES. vii LIST OF TABLES Page Table 2.1 The type of diseases , prevalence and the morbidity of 9 diseases in Malaysia Table 2.2 The number of dengue cases and death(s) reported from 15 2013 until 2015 Table 2.3 Dengue cases in Negeri Sembilan according to district 16 Table 2.4 The Number of Plasmodium knowlesi cases and its 21 prevalence Table 3.1 The PCR Master mix for the amplification of Dengue 85 virus sequence (cDNA synthesis before optimizing) Table 3.2 The PCR parameters for cDNA amplification of the 86 dengue virus complex Table 3.3 Mixture of PCR product after optimization Optimized 88 dengue virus complex with specific PCR master mix Table 3.4 PCR (standard) amplification condition after optimization. 89 To optimize dengue specific virus PCR condition Table 3.5 Mixture of PCR product before Optimization Process to 93 perform the Plasmodium spp. Table 3.6 PCR (standard) amplification condition before 94 optimization process to perform the Plasmodium species Table 3.7 Mixture of PCR master mix after optimization for 96 Plasmodium species to specific species (Plasmodium knowlesi ) sequence amplification Table 3.8 PCR (standard) amplification condition after optimization 97 viii for amplication of Plasmodium knowlesi sequence Table 3.9 The specific primer used in the sequence series 100 Table 4.1 Total number of whole blood samples collected for 104 dengue serology test and Blood Film Malaria/Filaria Parasite (BFMP) done from year 2012 until year 2014 Table 4.2 The number of patients who have seen by Medical Officer 106 in Hospital Tampin to investigate infected patients by vector-borne diseases from 2012 until 2014 Table 4.3 The Major Ethnic groups involved and positive in 107 dengue serology test Table 4.4 Identification of strain using BLAST analysis 112 Table 4.5 The mukims affectd within the number of dengue 114 infection cases from 2012 to 2014 Table 4.6 Identification of strain using BLAST analysis 118 Table 4.7 The Classic symptoms occurs in Dengue Patients 121 Table 4.8 The platelet count in dengue and malaria patients after 122 confirmed in dengue serology test and Blood Film Malaria/Filaria Parasite Table 4.9 The platelet count in dengue and malaria patients after 123 confirming with PCR Table 4.10 The records of 24 hours temperature taken from 126 Gemencheh Station as representive of the Tampin District temperature Table 4.11 The records of 24 hours temperature taken from 126 Gemencheh station as representive of the Tampin District ix temperature Table 4.12 The records of 24 hours mean relative humidity which 127 have been taken from Gemencheh station to representive the Tampin District humidity Table 4.13 The records of rainfall amount which have been taken 128 from Gemencheh station to representive the Tampin District temperature x LIST OF FIGURES
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