Detection and characterisation of Ureaplasma spp in men with and without urogenital symptoms NONTUTHUKO EXCELLENT MANINGI Detection and characterisation of Ureaplasma spp in men with and without urogenital symptoms by NONTUTHUKO EXCELLENT MANINGI Submitted in partial fulfilment of the requirements for the degree Magister Scientiae Department of Medical Microbiology Faculty of Health Sciences University of Pretoria Pretoria South Africa January 2012 The things that will destroy us are: politics without principle, pleasure without conscience, wealth without work, knowledge without character, business without morality, science without humanity, and worship without sacrifice. M ahatma G andhi Declaration I, Nontuthuko Excellent Maningi, hereby declare that the work on which this dissertation is based, is original and that neither the whole work nor any part of it has been, is being, or is to be submitted for another degree at this or any other university or tertiary education institution or examination body. ................................................................. Signature of candidate ............................................................... Date ACKNOWLEDGEMENTS First, I want to thank the Almighty, who made all things possible for me. To my family, especially my mother and my late father who never gave up on me, I wouldn’t have done it without their support and encouragement. I want to thank my supervisor Dr Kock for all the assistance, guidance and encouragement she has given me throughout my MSc. A special thanks to my co-supervisor Prof AA Hoosen, he was not only a supervisor but also a father to me, he has inspired me from the first day I met him. I also want to thank the Department of Medical Microbiology, Dr Adam and the NHLS (Tshwane Academic Division) for the opportunity to perform this research and use of their facilities. I would like to thank my colleagues from the Department of Medical Microbiology for all their help, and putting a smile on my face every day. I wish to acknowledge the financial grant for this study from the University of Pretoria. A special thanks to the Medical Research Council (MRC), National Research Foundation (NRF) and University of Pretoria for financial assistance received during my MSc study TABLE OF CONTENTS Page LIST OF FIGURES................................................................................................................iv LIST OF TABLES..................................................................................................................vi LIST OF ABBREVIATIONS...............................................................................................vii LIST OF ARTICLES SUBMITTED FOR PUBLICATION AND CONFERENCE CONTRIBUTIONS..................................................................................................................ix SUMMARY…...........................................................................................................................x CHAPTER 1: INTRODUCTION……………………………………………………………1 CHAPTER 2: LITERATURE REVIEW……………………………………………………5 2.1 Introduction .......................................................................................................................................... 5 2.2 History of Ureaplasma species ............................................................................................................ 6 2.3 Epidemiology of Ureaplasma species ................................................................................................. 7 2.4 Characteristics of Ureaplasma species ................................................................................................ 8 2.4.1 Scientific classification of the Ureaplasma species ............................................................ 8 2.4.2 Genomics of the genus Ureaplasma ................................................................................. 10 2.4.3 Morphological and physiological characteristics of the Ureaplasma species .................. 15 2.4.3.1 Synthesis of nucleic acids in Ureaplasma species.................................................... 15 2.4.3.2 The metabolic pathway of the Ureaplasma species.................................................. 16 2.4.3.2.1 Ammonium transporters ................................................................................ 19 2.4.3.2.2 F-ATPase complex of transporters ................................................................ 19 2.4.3.2.3 P-ATPase type cation transporters ................................................................. 19 2.4.3.2.4 Voltage-gated ion channel ............................................................................. 19 2.4.3.2.5 Magnesium ion transporter ............................................................................ 20 2.4.3.2.6 Amino acid-polyamine-organocation transporters......................................... 20 2.4.3.2.7 Multiple-oligosaccharidyl-lipid-polysaccharide family ................................. 20 2.4.3.2.8 Phosphoenolpyruvate-dependent sugar phosphotransferase transport system 20 2.4.3.2.9 Iron transporters ............................................................................................. 20 2.5 Pathogenesis and virulence of Ureaplasma infections ....................................................................... 21 2.6 Host defence mechanism to Ureaplasma species .............................................................................. 22 2.7 Mode of transmission of genital Ureaplasma species........................................................................ 22 2.8 Clinical manifestation of Ureaplasma species ................................................................................... 22 i 2.9 Laboratory detection of Ureaplasma species ..................................................................................... 24 2.9.1 In vitro growth of Ureaplasma species ............................................................................ 25 2.9.2 Commercial kits for the in vitro growth of Ureaplasma species ...................................... 26 2.9.3 Immunotyping of Ureaplasma species ............................................................................. 28 2.9.4 Molecular detection and characterisation assays for the genus Ureaplasma .................... 28 2.9.4.1 Conventional PCR assays for the detection of Ureaplasma species......................... 29 2.9.4.2 Real-time PCR assays for Ureaplasma species detection......................................... 29 2.10 Treatment of Ureaplasma species ...................................................................................................... 31 2.11 Antimicrobial susceptibility tests for Ureaplasma species ................................................................ 32 2.12 Antimicrobial drug resistance ............................................................................................................ 33 2.13 Prevention and control of Ureaplasma species .................................................................................. 34 2.14 Summary ............................................................................................................................................ 34 2.15 References .......................................................................................................................................... 35 CHAPTER 3: Characterization of Ureaplasma parvum Isolates from the Urethra of Men Attending a Family Practice 47 3.1 ABSTRACT ....................................................................................................................................... 47 3.2 INTRODUCTION ............................................................................................................................. 49 3.3 MATERIALS AND METHODS ....................................................................................................... 51 3.3.1 Study population and clinical specimens .......................................................................... 51 3.3.2 Cultivation of Ureaplasma species ................................................................................... 51 3.3.3 Antimicrobial susceptibility testing of Ureaplasma species............................................. 52 3.3.4 Total genomic DNA extraction ........................................................................................ 53 3.3.5 Real-time PCR for the detection of U. urealyticum .......................................................... 53 3.3.6 Multiplex Real-time PCR assays for the detection of U. parvum serovars....................... 54 3.3.7 Statistical analysis ............................................................................................................ 55 3.4 RESULTS .......................................................................................................................................... 56 3.4.1 Culture for the detection of Ureaplasma species .............................................................. 56 3.4.2 Susceptibility testing results ............................................................................................. 56 3.4.3 Real-time PCR detection of U. urealyticum ..................................................................... 57 3.4.4 Multiplex TaqMan real time PCR for the detection of U. parvum ................................... 58 3.4.5 Sensitivity and specificity of the real-time PCR assays for the detection of ureaplasmas 59 3.5 DISCUSSION ...................................................................................................................................