Fall 08 Molecular Studies of Treponema pallidum Craig Tipple Imperial College London Department of Medicine Section of Infectious Diseases Thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy of Imperial College London 2013 1 Abstract Syphilis, caused by Treponema pallidum (T. pallidum), has re-emerged in the UK and globally. There are 11 million new cases annually. The WHO stated the urgent need for single-dose oral treatments for syphilis to replace penicillin injections. Azithromycin showed initial promise, but macrolide resistance-associated mutations are emerging. Response to treatment is monitored by serological assays that can take months to indicate treatment success, thus a new test for identifying treatment failure rapidly in future clinical trials is required. Molecular studies are key in syphilis research, as T. pallidum cannot be sustained in culture. The work presented in this thesis aimed to design and validate both a qPCR and a RT- qPCR to quantify T. pallidum in clinical samples and use these assays to characterise treatment responses to standard therapy by determining the rate of T. pallidum clearance from blood and ulcer exudates. Finally, using samples from three cross-sectional studies, it aimed to establish the prevalence of T. pallidum strains, including those with macrolide resistance in London and Colombo, Sri Lanka. The sensitivity of T. pallidum detection in ulcers was significantly higher than in blood samples, the likely result of higher bacterial loads in ulcers. RNA detection during primary and latent disease was more sensitive than DNA and higher RNA quantities were detected at all stages. Bacteraemic patients most often had secondary disease and HIV-1 infected patients had higher bacterial loads in primary chancres. Treatment kinetics following benzathine penicillin injection were assessed in four men. The mean half-life for both blood and ulcer T. pallidum nucleic acid clearance was found to be short. All patients had serology consistent with cure at one month. Two T. pallidum strain types were found in Colombo, neither harbouring macrolide resistance. In London, several strain types were identified, the majority of which contained genetic determinants of macrolide resistance. 2 Declarations The microbiology departments at St Mary’s and Charing Cross Hospitals conducted laboratory tests used for the diagnosis of syphilis, including tests on cerebrospinal fluid. All clinical diagnoses referred to in this thesis were made by other physicians and not by myself. Unless otherwise stated or referenced, all other work presented in this thesis is my own. ---------- Craig Tipple The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work 3 Acknowledgements Firstly, I must thank my supervisors Dr Graham Taylor and Professor Myra McClure. Myra, I have valued highly your experience, support and your keen eye for detail (and grammar!). Graham, you have championed this project from almost the first time we met, and I am so very grateful for the time and the faith that you have invested me. I would like to thank Professors Sheila Lukehart and Cathy Ison for sharing their expertise of laboratory-based and clinical syphilis research and for allowing me to spend time in their laboratories to learn in vitro and animal techniques relevant to this project. I also thank my colleagues, many of whom are now friends, in the Jefferiss Trust Research Laboratories. Without your support, both practical and emotional, this work would not have been possible. I would like to thank especially Drs Steve Kaye and Maria-Antoinetta Demontis in this regard. Maria, you provided me an introduction to laboratory work with patience and kindness that I shall not forget. Steve, I cannot count the times that I wandered into your office with a question or query – you are a fount of knowledge, and I am most grateful for your support. I must also thank the patients and staff of both the Jefferiss Wing, St Mary’s Hospital and the sexual health clinics of Chelsea and Westminster Hospital for participating in the clinical studies presented in this thesis. Moreover, the staff of the McMichael-Wellcome Trust Clinical Research Facility at Hammersmith hospital and South Green ward at Charing Cross Hospital for hosting the inpatient element of the clinical study. I particularly thank Ms Lydia Hodson, Dr Rachael Jones, Sister Rosalind Nzeadi and Dr Karen Moseley for your kind assistance. Finally, this work would not have been at all possible without the generous support of the Mason Medical Foundation, The National Institute for Health Research and the NIHR Imperial College Biomedical Research Centre. Tim, Mum and Dad: I could not have done this without you. 4 Table of Contents Table of Contents ..................................................................................................................... 5 List of Tables ................................................................................................................................................. 6 List of Figures and Appendices............................................................................................................... 8 List of Abbreviations ............................................................................................................................... 10 Chapter 1 ................................................................................................................................. 11 Introduction ............................................................................................................................................... 11 Chapter 2 ................................................................................................................................. 58 Materials and Methods ........................................................................................................................... 58 Chapter 3 ................................................................................................................................. 90 Cross-sectional studies of T. pallidum nucleic acid quantification in vivo ........................... 90 Chapter 4 ............................................................................................................................... 177 The kinetics of early Syphilis treatment in vivo .......................................................................... 177 Chapter 5 ............................................................................................................................... 233 Molecular characterisation of T. pallidum strains ..................................................................... 233 Chapter 6 ............................................................................................................................... 260 Discussion ................................................................................................................................................. 260 References ............................................................................................................................. 271 Appendices ............................................................................................................................ 294 5 List of Tables Table 1.1 Characteristics of three members of the genus Treponema ..................................... 13 Table 1.2 Clinical manifestations of secondary syphilis .......................................................... 30 Table 1.3 Samples found to contain T. pallidum DNA ............................................................ 41 Table 2.1 Plasmids constructed for the project ........................................................................ 75 Table 3.1 A comparison of two methods for T. pallidum DNA extraction from Snostrips ..... 93 Table 3.2 T. pallidum RNA extraction from blood .................................................................. 96 Table 3.3 T. pallidum RNA extraction from blood .................................................................. 98 Table 3.4 Inter-assay variance of tpp047 qPCR ..................................................................... 102 Table 3.5 Inter-assay variation of the tpp047 qPCR .............................................................. 106 Table 3.6 Intra-assay variation of the tpp047 qPCR .............................................................. 107 Table 3.7 Specificity of three T. pallidum qPCRs .................................................................. 111 Table 3.8 Determination of the optimal assay for T. pallidum RT-qPCR ............................. 112 Table 3.9 Limiting dilution assay to quantify p16SRNA....................................................... 116 Table 3.10 Optimisation of T7 polymerase reaction conditions ............................................ 119 Table 3.11 Comparison of T7 RNA polymerases .................................................................. 120 Table 3.12 Intra-assay and inter-assay variability of 16S rRNA quantification .................... 125 Table 3.13 Comparison T. pallidum