Use of Molecular and Bioinformatics Tools for Developing Methods of Epidemiological Identification of Trypanosomes By Gaber Adwik Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy Jan 2016 College of science &Technology School of Environment & Life Sciences University of Salford I Contents List of Figures .............................................................................................................. V List of Tables ............................................................................................................ VIII Acknowledgment ........................................................................................................ IX Declaration Form ......................................................................................................... X Abbreviations ............................................................................................................... X Abstract ...................................................................................................................... XI Chapter 1 Introduction ............................................................................................... 13 1.1 History of Human Sleeping Sickness (HAT) .................................................. 14 1.2 The Pathogenic Parasite- Trypanosoma brucei ............................................ 15 1.3 Life Cycle of T. brucei ................................................................................... 18 1.4 Transmission Cycle ....................................................................................... 20 1.5 The vector- Tsetse fly .................................................................................... 21 1.6 Epidemiology of African sleeping sickness ................................................... 23 1.6.1 Sleeping sickness in Angola ........................................................................ 29 1.6.2 Current situation of HAT .............................................................................. 34 1.7 Diagnosis of Trypanosomiasis ...................................................................... 36 1.7.1 Parasitological Diagnosis ............................................................................. 37 1.7.2 Card Agglutination Test for Trypanosomiasis .............................................. 38 1.7.3 Molecular Diagnosis .................................................................................... 40 1.7.3.1 Genetic Markers ....................................................................................... 43 1.7.3.1 Internal Transcribed Spacer (ITS)-PCR .................................................... 47 1.7.3.1 Mini- And Micro-Satellite Markers ............................................................. 49 1.7.3.1 Random Amplified Polymorphic DNA (RAPD) .......................................... 49 1.7.3.1 Amplified fragment length polymorphism (AFLP) ..................................... 50 1.7.3.2 Mobile Genetic Element PCR (MGE-PCR) ............................................... 51 1.7.3.4 Loop-Mediated Isothermal Amplification (LAMP) ...................................... 52 1.8 Treatment of HAT .......................................................................................... 54 1.9 Aims .............................................................................................................. 52 Chapter 2 Materials and Methods .............................................................................. 53 2.1 Sample collection ....................................................................................... 54 2.2 Sample Processing .................................................................................... 54 2.3 DNA Chelex extraction ............................................................................... 55 II 2.4 Trypanosome DNA serial dilutions ............................................................. 55 2.5 PCR Amplification ...................................................................................... 55 2.6 Visualization ............................................................................................... 58 2.7 DNA extraction from agarose gels ............................................................. 58 2.8 Sequencing of the PCR product (DNA) ...................................................... 58 2.9 Restriction enzymes digestion.................................................................... 59 2.10 Bioinformatics and analysis of T.brucei genome TREU927 ....................... 59 Chapter 3 Identification of African trypanosome species from FTA card using ITS-PCR ............................................................................................................................ 61 3.1 Introduction ................................................................................................ 61 3.1.1 Aims and Objectives .................................................................................... 66 3.2 Methods ..................................................................................................... 67 3.3 Results ....................................................................................................... 68 3.3.1 Description of the samples of infected blood immobilised on FTA cards from human trypanosomiasis patients in Angola .......................................................... 68 3.3.2 DNA amplification and diagnosis of trypanosomiasis .................................. 74 3.3.2.1 Sensitivity of nested ITS-PCR .................................................................. 74 3.3.3 Identification of nested ITS-PCR amplified products (DNA sequencing) ...... 90 3.3.4 Analysis of positive and negative infections in the Angolan samples ........... 95 3.4 Discussion ....................................................................................................... 99 Chapter 4 . Identification of trypanosomes from FTA cards spotted with blood using MGE-PCR.......................................................................................................... 107 4.1 Introduction .............................................................................................. 107 4.2 Objectives ................................................................................................ 113 4.3 Methods ................................................................................................... 114 4.4 Results ..................................................................................................... 115 4.4.1 Development of the use of MGE-PCR in FTA cards ................................ 115 4.4.2 Chelex-MGE-PCR detection of trypanosomes in FTA cards .................... 117 4.4.3 Investigation of REV B specificity using DNA sequencing to confirm band sizes ......................................................................................................... 123 4.4.4 Improving DNA sequencing of REV B amplified products using restriction enzyme digestion to semi-purify fragments .............................................. 124 4.4.5 Negative control amplification by REV B ................................................... 129 4.4.6 Alternative approach to improve DNA sequencing data ............................ 130 4.4.7 Post RIME A and RIME B amplification analysis (DNA sequencing) ......... 132 III 4.5 Discussion .................................................................................................... 139 Chapter 5 Investigation of Mobile Genetic Elements Found Inserted in Retrotransposon Hotspot Genes (RHS) in the T. brucei TREU927 Genome .......... 143 5.1 Introduction ................................................................................................. 143 5.2 Objectives ................................................................................................... 149 5.3 Methods ...................................................................................................... 150 5.4 Results ........................................................................................................ 151 5.4.1 Identification of retrotransposon hot spot (RHS) genes in the TREU927 genome .............................................................................................................. 151 5.4.2 Evolutionary relationship of RHS genes: .................................................. 155 5.4.2.1 RHS 3 and RHS4 mutational change analysis ...................................... 158 5.4.3 Evolutionary time dimensions of RHS genes ........................................... 166 5.4.4 Mobile genetic element markers in the differentiation of trypanosome strains ................................................................................................................. 169 5.4 Discussion .................................................................................................... 179 6.1 General discussion ....................................................................................... 184 6.1 Future work ................................................................................................. 198 6.2 Conclusion .................................................................................................. 199 7. References ........................................................................................................ 200 Appendices .............................................................................................................