Investigating the 2005 singaporean dengue outbreak

INVESTIGATING THE 2005 SINGAPOREAN DENGUE OUTBREAK LUKAS TANNER B.Sc (Major in Molecular Biology), University of Basel A THESIS SUBMITTED FOR THE DEGREE OF A MASTER OF SCIENCE IN INFECTIOUS DISEASES, VACCINOLOGY & DRUG DISCOVERY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE & BIOZENTRUM UNIVERSITY OF BASEL 2007 Acknowledgments I would like to thank my supervisor Mark Schreiber for his patience and support during my masters project His guidance made my time at NITD a scientific and educational experience which is invaluable for my future career I also thank Liu Wei for taking the time to introduce me to virus work and to the sequencing project He provided great support during the vicious times of PCR optimization I would also like to thank Subhash Vasudevan for his fair comments on the project and for giving me the opportunity to pursue my masters project at NITD I am also very grateful to Feng Gu, Katja Fink and Cedric Ng who critically read the manuscript and provided me with the necessary amendments I also would like to mention Robert Zweighardt who took the time to give suggestions from a non-dengue perspective From GIS, I thank Pauline Aw Poh Kim for her part in the sequencing Without her effort it would not have been possible to sequence the EDEN isolates In particular, I am grateful to Ong Swee Hoe for his phylogenetic analyses and for his helpful discussions with regard to sequence alignments He provided me with the necessary background to be able to understand dengue virus phylogeny I additionally express my gratitude to Martin Hibberd for his advice on dengue immunology I express thanks to the whole EDEN team but especially, to Eng Eong Ooi who provided me with the clinical and immunological data It was a pleasure to collaborate with him and he gave me useful suggestions I wish to thank Reinhard Bergmann from NIBR who was helpful in the statistical analyses He provided me with the necessary knowledge and always had time to answer my many questions I would like to express my thanks to all the people in the dengue unit who made my time at NITD not only a scientific but also a personal experience In particular, I express my gratitude to Cedric, Viral, Sarah, Joanne, Katja, Indira, Anne, Dina, Mee, Cheryl, Celine, Alex, Jasmin and Selina who provided me with the necessary friendship and motivation It was nice to have Stevie and Tommy as flat mates and friends The time with all of you was great and hopefully we will meet someday, somewhere again… I am most grateful to my parents Suzanne and Marcel who gave me this unique opportunity to come to Singapore Their support in good but especially in difficult times is invaluable and I am happy to have them as parents A special thanks goes to my two sisters Sabine and Catherine Talking and spending time with them is great and gives me new energy to go on in my life Last but not least, I show gratitude to my friends back home in Switzerland who always had time to talk to me on the phone I’ve realized that without their good friendship I would not have the energy to fulfill my goals…Merci vielmol! Table of Contents Acknowledgments .2 Table of Contents Summary 10 List of Tables .12 List of Figures 18 List of Abbreviations 22 Introduction 24 1.1 Epidemiology of Dengue 25 1.1.1 The Global Emergence of Dengue 25 1.1.1.1 Situation in the Americas 26 1.1.1.2 Situation in the Asia/Pacific 27 1.1.1.3 Reasons for the Global Emergence of Dengue 28 1.1.2 Public Health, Social & Economic Impacts of Dengue 29 1.1.2.1 Impact on Public Health 29 1.1.2.2 Impact on Society 29 1.1.2.3 Impact on Economy 30 1.1.3 Epidemiological Situation in Singapore 31 1.1.3.1 Dengue Epidemiology in Singapore 31 1.1.3.2 The Early DENgue (EDEN) Study 33 1.2 Classical Dengue Fever, Dengue Haemorrhagic Fever & Dengue Shock Syndrome34 1.2.1 Clinical Manifestations of Dengue 34 1.2.1.1 Classical Dengue Fever 34 1.2.1.2 Dengue Hemorrhagic Fever and Dengue Shock Syndrome 35 1.2.2 Dengue Diagnosis and Discussion of the WHO Classification Scheme 36 1.2.2.1 Important Laboratory Tests for Dengue Diagnosis 36 1.2.2.2 WHO Classification Scheme 37 1.2.3 Prevention and Treatment of Dengue .39 1.2.3.1 Vector Control 39 1.2.3.2 Dengue Vaccines 39 1.2.3.3 Drugs against Dengue 40 1.2.3.4 Current Treatment of DF and DHF/DSS 40 1.3 The Causative Agent: Dengue Virus 42 1.3.1 Phylogeny 42 1.3.1.1 Dengue Serotype 43 1.3.1.2 Dengue Serotype 44 1.3.1.3 Dengue Serotype 45 1.3.1.4 Dengue Serotype 46 1.3.1.5 Serotype Switch & Clade Replacement 47 1.3.2 Dengue Virus Lifecycle 48 1.3.2.1 Structure of Dengue Virions 48 1.3.2.2 Viral Entry 50 1.3.2.3 Viral Replication, Assembly and Exocytosis 51 1.3.2.4 Extrinsic versus Intrinsic Lifecycle 53 1.4 Immunology of Dengue Virus Infections 56 1.4.1 1.5 Dengue Pathogenesis and Host Immune Response 56 1.4.1.1 Early Events in the Host after Infection 56 1.4.1.2 Important Mediators of Innate Immunity during Infection 57 1.4.1.3 Antibody-Dependent Enhancement (ADE) 57 1.4.1.4 Differences in Secondary T-Cell Responses 58 1.4.1.5 Important Cytokines during Infection 60 1.4.1.6 DHF/DSS: An Immune-Mediated Machanism 60 1.4.1.7 Viral Determinants and Disease Outcome 61 Aims of the Studies 64 Materials & Methods .65 2.1 Analysis of Clinical Data 66 2.1.1 Collection and Preprocessing of Clinical Data 66 2.1.1.1 Clinical Data used for Analyses 67 2.1.1.2 Clinical Data used for Severity Modeling 68 2.1.1.3 Immunological and Clinical Data used for Analyses 69 2.1.1.4 Summary of Clinical and Immunological Data used for Analyses 70 2.1.2 Statistical Analyses used for the Description of Clinical and Immunological Data…… 73 2.1.2.1 Univariate Analyses 73 2.1.2.2 Multivariate Analyses 74 2.1.3 Decision Tree Analyses for Disease Modeling .74 2.1.3.1 Classifier Modeling 77 2.1.3.2 Classifier Evaluation 78 2.1.3.3 Epidemiological Analyses of Parameters included into the Models 81 2.1.3.4 Summary of generated Models included in the Thesis 82 2.2 Full Length Genome Sequencing of Dengue Virus Isolates 83 2.2.1 Preparative RT-PCR Step 83 2.2.1.1 Propagation of Virus 84 2.2.1.2 Quantification of Virus by Plaque Assay 84 2.2.1.3 Extraction of viral RNA and cDNA Synthesis 85 2.2.1.4 Amplification of cDNA by Polymerase Chain Reaction 87 2.2.2 Phylogenetic Analyses of Dengue Virus Genomes 90 2.2.2.1 Sequencing Process, Assembly and Quality Control 90 2.2.2.2 Sequence Alignment and Phylogentic Analyses 90 Results .91 3.1 Decision Tree Analyses of Clinical Data 92 3.1.1 Distinguishing Dengue Fever from other febrile Illnesses .92 3.1.1.1 Dengue Prediction based on Clinical Data 92 3.1.1.2 Dengue Prediction based on Cytokine and Clinical Data 97 3.1.1.3 Dengue Prediction based on Cytokine Data 108 3.1.2 Prediction of Disease Severity in Dengue Patients 114 3.1.2.1 Prediction of Hospitalization based on Clinical Data 114 3.1.2.2 Prediction of Hospitalization based on Cytokine and Clinical Data 117 3.1.2.3 Prediction of Hospitalization based on Cytokine Data 120 3.1.2.4 Using a Platelet Count [...]... Introduction 1 Introduction Investigating the 2005 Singaporean Dengue Outbreak 1.1 Epidemiology of Dengue 1.1.1 The Global Emergence of Dengue Dengue is an old and the most important arthropod-borne viral disease affecting humans in terms of morbidity There are reports in the medical literature about epidemics caused by an illness comparable to dengue that reach back to the late 17th century The Chinese, however,... Introduction 1.2.2 Investigating the 2005 Singaporean Dengue Outbreak Dengue Diagnosis and Discussion of the WHO Classification Scheme 1.2.2.1 Important Laboratory Tests for Dengue Diagnosis Besides the description of clinical symptoms, there are also clinical laboratory tests that are useful in the diagnosis of dengue These clinical tests include a complete blood cell count (CBC), especially the white blood... highly 26 1 Introduction Investigating the 2005 Singaporean Dengue Outbreak acceptable effect on combating dengue fever It resulted in the declining or even disappearance of DF throughout the region (Graham et al., 1999; Gubler, 2004) 1.1.1.2 Situation in the Asia/Pacific However, in the Asia/Pacific region, DF was a common occurrence in the first 50 years of the 20th century There was an epidemic every... Introduction 1.1.2 Investigating the 2005 Singaporean Dengue Outbreak Public Health, Social & Economic Impacts of Dengue 1.1.2.1 Impact on Public Health Due to poor dengue surveillance and due to great similarities in disease manifestation to other tropical diseases such us malaria and chikungunya fever (Rigau-Perez et al., 1998), the early stages of dengue outbreaks are mostly not detected and thus, dengue cases... Americas were highly successful resulted in the decrease of public awareness The global spread of dengue disease was mainly ignored by public health 29 1 Introduction Investigating the 2005 Singaporean Dengue Outbreak officials and hence, the people had to tolerate the interruption of their daily life by epidemics occurring every few years (Gubler, 2002) Especially, the insufficient surveillance and case... between 2004 -2005 In 2005, Singapore reported an increase of more than 50% cases compared to 2004 (MOH, 2006) 31 1 Introduction Investigating the 2005 Singaporean Dengue Outbreak In Singapore, however, young male adults have the highest risk of being infected with dengue virus (Ooi et al., 2003; Ooi et al., 2006; Wilder-Smith et al., 2004) In 2005, the male to female ratio was 1.4:1 with the highest... be found 32 1 Introduction Investigating the 2005 Singaporean Dengue Outbreak 1.1.3.2 The Early DENgue (EDEN) Study To address this goal, a longitudinal study has been implemented by a joint effort between the Tan Tock Seng Hospital (TTSH), the Genome Institute of Singapore (GIS), the Novartis Institute for Tropical Diseases (NITD), the Environmental Health Institute (EHI), the Singapore Tissue Network... Correlate virus virulence with their sequences and their replication properties and (6) Refine early dengue clinical and laboratory diagnostic tools (Low et al., 2006) The study has been launched in 2005 and during the 2005 Singaporean dengue outbreak, 455 patients were enrolled and 133 of them were PCR confirmed dengue positive cases An initial blood and saliva sample is taken on the first visit (1 to 3... multiple dengue virus serotypes (Gubler, 2004) This resulted in an increased transmission of multiple serotypes and in the sequential emergence of dengue hemorrhagic fever (DHF) in the 1950s with a first outbreak in Manila, Philippines Singapore experienced its first DHF epidemic in the early 1960s (Gubler, 2004) 27 1 Introduction Investigating the 2005 Singaporean Dengue Outbreak 1.1.1.3 Reasons for the. .. / Dengue Shock Syndrome (DHF/DSS) and more than 20,000 deaths occur per year (WHO, 2002) In the last 25 years of the 20th century, dengue has emerged as a major public health problem and epidemics have a tremendous impact on social as well as economic structures of society especially in developing countries of the tropics (Gubler, 2002) 25 1 Introduction Investigating the 2005 Singaporean Dengue Outbreak ... 23 Introduction Introduction Investigating the 2005 Singaporean Dengue Outbreak 1.1 Epidemiology of Dengue 1.1.1 The Global Emergence of Dengue Dengue is an old and the most important arthropod-borne... Introduction Investigating the 2005 Singaporean Dengue Outbreak 1.2.2.2 WHO Classification Scheme The WHO scheme for dengue diagnosis consists of the above mentioned dengue case definition (having dengue. .. lengths and therefore suggesting that viruses 43 Introduction Investigating the 2005 Singaporean Dengue Outbreak of this genotype are mixed regardless of their location On the other hand, the picture