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University of Ghana College of Basic and Applied Sciences by Shirley Victoria Simpson (10551058) This Thesis Is Submitted To

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University of Ghana College of Basic and Applied Sciences by Shirley Victoria Simpson (10551058) This Thesis Is Submitted To UNIVERSITY OF GHANA COLLEGE OF BASIC AND APPLIED SCIENCES ISOLATION AND CHARACTERIZATION of Haemophilus ducreyi STRAINS FROM CHILDREN WITH CUTANEOUS LESIONS IN YAWS ENDEMIC REGIONS, GHANA BY SHIRLEY VICTORIA SIMPSON (10551058) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL MOLECULAR CELL BIOLOGY OF INFECTIOUS DISEASES DEGREE JULY, 2017 DECLARATION This is to certify that this thesis is the result of research undertaken by me, Shirley Victoria Simpson towards the award of Master of Philosophy in Molecular Cell Biology of Infectious Diseases in the Department of Biochemistry, Cell and Molecular Biology, School of Biological Sciences, College of Basic And Applied Sciences, University of Ghana. Signature--------------------------------------- Date-------------------------- Shirley Victoria Simpson (Candidate) Signature--------------------------------------- Date----------------------- Prof. Kennedy Kwasi Addo (Supervisor) Signature------------------------------------ Date----------------------- Dr. Lydia Mosi (Co-Supervisor) i ABSTRACT Recent discovery of cutaneous H. ducreyi has complicated the epidemiology of Yaws in endemic countries. Yaws and H. ducreyi ulcers are clinically indistinguishable from each other and some other causes of skin ulcerations. The aim of the study was to isolate and characterize H. ducreyi strains from lesions of children in yaws-endemic areas. Symptomatic patients were first screened with Dual Path Platform (DPP-RDT) Syphilis Screen & Confirm test kit (Chembio, Medford, New York) for yaws. Lesion exudates were tested by culture for H. ducreyi and real-time multiplex PCR assays were used to identify T.p subsp. pertenue DNA and H. ducreyi DNA. Azithromycin (AZT) resistance markers were screened for in T.p subsp. pertenue PCR positives. Bacterial 16S rRNA gene was amplified and sequenced to detect the presence of other pathogenic bacteria. Patient data showed 84 of 115 more males than females, mean aged 10 years. Eighty-seven percent had a clinically apparent skin lesion with few having skin conditions clinically consistent with yaws. Dual DPP-RDT positives were 64 while dual DPP-RDT negatives were 51. Of 60 bacteria culture positives obtained from symptomatic patients, 7 yielded a definitive diagnosis of H. ducreyi. Isolated cutaneous H. ducreyi strains by their appearance in colony morphology and colour differed compared to genital H. ducreyi strain, HD 35000. Out of 112 samples, 32 were H. ducreyi PCR positive, 11 were T.p subsp. pertenue PCR positive, while 1 had both pathogens. An A2058G point mutation in the 23S rRNA gene of T.p subsp. pertenue indicates resistance to AZT. A total of 69 of 112 samples with unknown aetiology could possibly be any of these bacterial species; Fusobacterium necrophorum subsp. funduliforme, Catonella morbi and Staphylococcus capitis subsp. capitis amongst others. This study suggest the need to isolate bacterial species associated with cutaneous lesions to screen for other antibiotics to be used as a combination therapy with AZT during mass drug administration (MDA) activities. ii DEDICATION To God Almighty, all Glory to His Holy name and my son, Ashley Drew Dake. iii ACKNOWLEDGEMENTS I wish to express my deep appreciation and indebtedness to my supervisors, Prof. Kennedy Kwasi Addo and Dr. Lydia Mosi for their valuable guidance, encouragement, and support during this MPhil program. I wish to express my heartfelt gratitude to Dr. Kingsley Asiedu, from WHO for his immense support and encouragement throughout my MPhil study. I would like to show appreciation and acknowledge Dr. Cheng Chen, Dr. Allan Pillay, Dr. Samantha Katz and Kai-Hua Chi all of CDC, USA for the technology transfer and donation of laboratory reagents. I wish to acknowledge WACCBIP (West African Center for Cell Biology of Infectious Pathogens) for the second year scholarship and the platform to showcase my findings. I would also like to show appreciation to Dr. Gloria Ivy Mensah, Prof. William Ampofo, Dr. Cynthia Kwakye-Maclean for their diverse roles they played, not forgetting all members of the Bacteriology and Virology Department of NMIMR. I wish to thank Mr. Abiola Isawumi and Ms. Adisa Abass for immense laboratory support for this work. Special thanks to Ms. Ivy Amanor and Ms. Stephanie Clement-Owusu. Special acknowledgements and appreciation go to my family, Mr. Paul Kow Simpson, Ms. Grace Felicity Opoku, Mr. Richard Cudjoe, Mr. Francis Willie Laast and my younger siblings for their prayers and support both financially and physically. iv TABLE OF CONTENTS DECLARATION ................................................................................................................... i ABSTRACT .......................................................................................................................... ii DEDICATION .................................................................................................................... iii ACKNOWLEDGEMENTS ................................................................................................. iv TABLE OF CONTENTS ...................................................................................................... v LIST OF TABLES ............................................................................................................ viii LIST OF FIGURES ............................................................................................................. ix CHAPTER ONE ................................................................................................................... 1 INTRODUCTION ................................................................................................................ 1 1.1 Rationale ...................................................................................................................... 4 1.2 Aim of the Study ......................................................................................................... 6 1.3 Specific objectives ....................................................................................................... 7 CHAPTER TWO .................................................................................................................. 8 LITERATURE REVIEW...................................................................................................... 8 2.1 History of Chancroid ................................................................................................... 8 2.2 The genus Haemophilus .............................................................................................. 9 2.3 Characteristics of Haemophilus ducreyi .................................................................... 10 2.3.1 Classes of Haemophilus ducreyi ......................................................................... 11 2.3.1.1 Subclades of Haemophilus ducreyi strains .................................................. 12 2.4 Pathogenesis of Chancroid ........................................................................................ 14 2.5 Clinical presentation of Chancroid ............................................................................ 15 2.6 Host immune response to Haemophilus ducreyi infection ........................................ 18 2.7 Epidemiology of Haemophilus ducreyi infections .................................................... 20 2.8 Yaws .......................................................................................................................... 23 2.8.1 The causative agent ............................................................................................. 24 2.8.2 Pathogenesis........................................................................................................ 25 2.8.3 Clinical features of Yaws in children ................................................................. 26 2.8.3.1 Primary Yaws ............................................................................................... 26 2.8.3.2 Secondary Yaws ........................................................................................... 27 2.8.3.3 Latent Yaws ................................................................................................. 29 2.8.3.4 Tertiary Yaws ............................................................................................... 29 2.8.4 Host immune response to Yaws infection .......................................................... 29 2.8.5 Epidemiology ...................................................................................................... 30 2.8.6 Diagnosis ............................................................................................................ 32 2.9 Laboratory methods for diagnosis of Haemophilus ducreyi infections..................... 34 2.9.1 Bacteriological diagnostic techniques ................................................................ 35 2.9.2 Mass spectrometric identification method .......................................................... 36 2.9.3 Serological detection........................................................................................... 37 2.9.4 Molecular-based detection .................................................................................. 37 2.10 Treatment ................................................................................................................. 38 v 2.11 Antimicrobial susceptibility patterns ......................................................................
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