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MYCO WELL D-ONE: Can a New Rapid Diagnostic Detect Ureaplasma Spp. and Mycoplasma Hominis in Genitourinary Medicine Samples?

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MYCO WELL D-ONE: Can a New Rapid Diagnostic Detect Ureaplasma Spp. and Mycoplasma Hominis in Genitourinary Medicine Samples? MYCO WELL D-ONE: Can a new rapid diagnostic detect Ureaplasma spp. and Mycoplasma hominis in genitourinary medicine samples? A thesis submitted in candidature for the degree of Master of Philosophy (MPhil) 2019 Daniel John Morris School of Medicine Summary of Thesis The genital mycoplasmas – Mycoplasma hominis, Ureaplasma parvum and Ureaplasma urealyticum – are frequently associated with symptomatic genitourinary medicine patients. Currently there is no provision to test for these organisms within the National Health Service. Ureaplasma spp. are isolated from 11–26% of men with non-gonococcal urethritis. Bacterial vaginosis (BV) – present in 20–30% of females attending – has been repeatedly linked to M. hominis. The MYCO WELL D-ONE is a rapid culture-based assay that utilises specialist media to detect these organisms in samples collected as part of routine sexual health screenings. Alongside detection, the MYCO WELL D-ONE concurrently screens organisms for antibiotic resistance. IRAS ethics number 230693 was granted for this study. 983 Samples were collected from 857 patients attending sexual health clinics in Cwm Taf: 528 female patients (121 provided both urine and swabs) and 329 male patients. This is the first clinical validation of the MYCO WELL D-ONE to be carried out in the UK against the current ‘gold standard’ molecular detection methods. Isolates highlighted as resistant by MYCO WELL D-ONE were subjected to confirmatory antibiotic susceptibility testing (AST) in accordance with the current CLSI guidelines. Isolates confirmed as resistance underwent whole genome sequencing to determine the underlying genetic mechanism conferring their resistance. Ureaplasma spp. was isolated from 57.2% of female patients compared to 21.6% of males. For M. hominis, 16.3% of females and 4.3% of males were infected. The MYCO WELL D-ONE displayed sensitivity values of 91.8% and 78.2%, alongside specificity values of 96.49% and 98.84%, for the detection of Ureaplasma spp. and M. hominis, respectively. Antibiotic resistance rates in Ureplasma spp. were found to be 0.54% for tetracycline and levofloxacin, with a tetracycline resistance rate of 1% for M. hominis. The prevalence of genital mycoplasma in the South Wales Valleys convergence area is significantly higher in the female population, consistent with other studies at different geographic locations. The MYCO WELL D-ONE is a simple, sensitive and specific means of detecting these organisms. It offers clinicians a rapid, easy-to-use and inexpensive method of determining the presence of mycoplasma in symptomatic sexual health patients. ix Daniel John Morris Declaration This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed ………………………………………… (candidate) Date ………………………… STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree of ………………………… (insert MCh, MD, MPhil, PhD etc, as appropriate) Signed ………………………………………… (candidate) Date ………………………… STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. The views expressed are my own. Signed ………………………………………… (candidate) Date ………………………… STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed ………………………………………… (candidate) Date ………………………… STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loans after expiry of a bar on access previously approved by the Academic Standards & Quality Committee. Signed ………………………………………… (candidate) Date ………………………… x Daniel John Morris Acknowledgements My deepest thanks to Dr Brad Spiller, to whom I am indebted a great a deal, for both the opportunity to carry out this study and for the knowledge and skills I gained under his supervision. A brilliant, charismatic and inspiring microbiologist whose enthusiasm for mycoplasma can only be described with one ironic adjective – infectious. I whole-heartedly appreciate the time and effort Dr Spiller invested in me and this project. His unique apprenticeship style of teaching and hands-on way of developing my research skills was hugely valued, providing both guidance and freedom in equal measure. I am also thankful for his unrivalled ability to dispel despair following unexpected experimental results with his trademark ‘Well, that’s interesting!’. To Dr Lucy Jones, thank you! I could not have asked for a more personable, friendly and gifted clinician from which to learn the clinical side of research. Dr Jones possesses an immense ability to cultivate a unique culture in her clinics, one that facilitates top-class patient care running seamlessly and synergistically alongside research activities. Your words of encouragement as I trundled out of Keir Hardie late on Thursday evenings, laden like a packhorse with samples, pipettes, media etc. spurred me on no end. Also, a huge thank you to all the research nurses and clinical staff at Dewi Sant, YCC and Keir Hardie GUM clinics – there are too many to list individually – I’m forever grateful for the cuppas and lunchtime chats. But I am ever more appreciative of the way in which you supported the project, your willingness to engage and persistence in sample collection are the reason so many were collected. My thanks also to Edward Portal for his help in sequencing my isolates and being so generous with your time whenever I needed to find anything. Personally, I’d like to thank my Mother, Father, Sister, Nan and Bamps, for without such a loving family behind me, completing this project would not have been possible. They have always believed in me, encouraged me, supported me and showed an interest in everything I do. I would also like to thank Brad Evans, Neil Williams and Russ Evans – my MMA and Brazilian Jiu Jitsu coaches – as well as all my training partners at Combat Training Centre. Without these men xi Daniel John Morris introducing me to Martial Arts I absolutely would not have had the confidence or determination to complete this degree whilst working and training. My friends (The Boys) – who are too numerous to name, but who know who they are – also played a huge role in supporting me through this degree. Thank you for showing an interest in this project and sharing a pint and a laugh with me when it was needed. Thanks also to my funders KESS2 and Health First Consulting, also thanks to CPM SAS for kindly providing the MYCO WELL D-ONE assays. Finally, thanks to my beautiful girlfriend Beth, for putting up with never seeing me as I worked 7- days-a-week throughout this project, we can now spend some time together and go on holidays. Thank you for your never-ending support, patience and tea-making. xii Daniel John Morris Abbreviations ATP Adenosine triphosphate AST Antimicrobial sensitivity testing bp Base pairs BPD Bronchopulmonary dysplasia BV Bacterial vaginosis C. trachomatis Chlamydia trachomatis CCU Colour changing unit CD Circular dichroism CDC The Centers for Disease Control and Prevention CDS Coding sequence CFU Colony forming unit CLSI Clinical and Laboratory Standards Institute CR Complement receptor CSF Cerebrospinal fluid DNA Deoxyribonucleic acid E. coli Escherichia coli G + C Guanine and Cytosine G. vaginalis Gardnerella vaginalis GAG Glycosaminoglycan G-CSF Granulocyte-colony stimulating factor HIV Human Immunodeficiency Virus HPV Human papilloma virus HRT Hormone replacement therapy xiii Daniel John Morris Ig Immunoglobulin IL Interleukin IS Insertion sequence IVF in vitro fertilisation Kda Kilodaltons M. fermentans Mycoplasma fermentans M. genitalium Mycoplasma genitalium M. hominis Mycoplasma hominis M. penetrans Mycoplasma penetrans M. pneumoniae Mycoplasma pneumoniae MAbs Monoclonal antibodies MAPK Mitogen-activated protein kinase MBA Multiple banded antigen M-CSF Macrophage-colony stimulating factor MHSM M. hominis-specific media MLSK Macrolides-lincosamides-streptogramin group and ketolides MMP Matrix metalloproteinase mRNA Messenger ribonucleic acid MSM Men who have sex with men MSW Men who have sex with women N. gonorrhoeae Neisseria gonorrhoeae N. meningitidis Neisseria meningitidis NF-KB Nuclear factor kappa-light-chain-enhancer of activated B cells NGU Non-gonococcal urethritis xiv Daniel John Morris NO Nitric oxide NPV Negative predictive value NSC Non-specific cervicitis NSU Non-specific urethritis Opp Oligopeptide permease ORF Open reading frame PAI Pathogenicity islands sterile saline Phosphate-buffered saline PCR Polymerase chain reaction PG Prostaglandin Pgk Phosphoglycerate kinase PHE Public Health England PID Pelvic inflammatory disease PPROM Preterm prelabour rupture of the membranes PPV Positive predictive value PRR Pattern recognition receptors PTB Preterm birth qPCR Quantitative polymerase chain reaction QRDR Quinolone resistance-determining region RNA Ribonucleic acid rRNA Ribosomal ribonucleic acid SPTL Spontaneous preterm labour STD Sexually transmitted disease STI Sexually transmitted infection xv Daniel John Morris SV Serovar T. vaginalis Trichomonas vaginalis TK Thymidine kinase TLR Toll-like receptor TNF- α Tumour necrosis factor-α tRNA Transfer ribonucleic acid U. parvum Ureaplasma parvum U. urealyticum Ureaplasma urealyticum
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