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Investigation of a New Focus of Cutaneous Leishmaniasis in Ghana PDF

236 Pages·2016·4.27 MB·English
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1 Investigation of a New Focus of Cutaneous Leishmaniasis in Ghana By Godwin Kwakye-Nuako BSc, MSc, MPhil PhD Thesis In Biomedical and Life Sciences Lancaster University Faculty of Health and Medicine Biomedical and Life Sciences February, 2016 2 DECLARATION I declare that the content of this thesis is my own work, except were clearly stated, and has not been submitted in substantially the same form for the award of a higher degree elsewhere. 3 DEDICATION To my wife and children: Charlotte Omane Kwakye-Nuako Nana Akua Pokua Kwakye-Nuako Mame Yaa Kwakye-Nuako Barima Yaw Kwakye-Nuako 4 ACKNOWLEDGEMENT My first and foremost thanks go to my supervisor, Prof. Paul Andrew Bates, for his extraordinary mentorship over the years. The sense of independence, confidence and tolerance you instilled in me will forever be treasured. Many thanks to Prof. Johnson Nyarko Boampong, Dr Elvis Ofori Ameyaw both of University of Cape Coast, Ghana and Dr Alison Beckett, University of Liverpool, for their technical support. I render my sincere thanks to the Leishmania Research Group, Lancaster University, UK, for their significant contribution. My special thanks to Michelle Bates and Dr Manal Jamjoom for coaching me in the laboratory techniques and immense support they gave me. I would also like to extend my gratitude to the staff of the Division of Biomedical and Life Sciences, Lancaster University, for their technical support during the work, as well as colleagues at Noguchi Memorial Institute for Medical Research, Ghana. I would like to express my gratitude to Amoako-Sakyi family, my mother Angelina Yaa Nkrumah, my father Eric Baah-Nuako, my grandmother Nana Ama Pokua Kobi and Rt. Rev. and Mrs Omane-Achamfuor, for all the support both spiritual and physical. Now my deepest appreciation to my wife Charlotte Omane Kwakye-Nuako for her unflinching morale, emotional, spiritual and physical support without which I would not have come this far. The work received financial support from the Lancaster University studentship and Wellcome Trust, UK. 5 TABLE OF CONTENTS DECLARATION i DEDICATION ii ACKNOWLEDGEMENT iii TABLE OF CONTENTS iv CONTENTS v REFERENCE viii APPENDIX viii LIST OF FIGURES ix LIST OF TABLES xii ABBREVIATIONS xiii ABSTRACT xvii 6 Contents Chapter One 1.0 General Introduction and Literature Review……………………………... 1 1.1 The public health importance of leishmaniasis…………………………... 1 1.2 Clinical forms of leishmaniasis…………………………………………... 4 1.2.1 Cutaneous leishmaniasis……………………………………………….. 5 1.2.2 Mucocutaneous leishmaniasis………………………………………….. 8 1.2.3. Visceral leishmaniasis…………………………………………………. 9 1.2.4 Leishmania-Human Immunodeficiency Virus co-infection……………. 11 1.3 Leishmania parasites and their life cycle………………………………… 14 1.4 Diagnosis and Leishmania species identification………………………… 22 1.5 Treatment of leishmaniasis………………………………………………. 24 1.6 The sand fly vectors of leishmaniasis……………………………………. 28 1.7 Emerging foci of the disease outside Africa……………………………... 33 1.7.1 Leishmaniasis in Australia……………………………………………... 33 1.7.2 Leishmaniasis in Thailand and the Caribbean…………………………. 37 1.7.3 Leishmaniasis in Sri Lanka…………………………………………….. 39 1.7.4 Leishmaniasis in Europe……………………………………………….. 42 1.8 Leishmaniasis in Africa…………………………………………………... 47 1.9 Leishmaniasis in Ghana………………………………………………….. 51 1.10 Rationale/Justification of the study…………………………………….. 53 1.11 Aims of the project……………………………………………………… 56 1.11.1 Specific objectives…………………………………………………….. 56 Chapter Two 2.0 General Materials and Methods………………………………………….. 57 2.1 Study Sites………………………………………………………………... 57 7 2.2 Study Participants………………………………………………………… 58 2.3 Case Search………………………………………………………………. 59 2.4 Sample collection………………………………………………………… 60 2.5 Culture of field isolates…………………………………………………... 61 2.6 Storage of samples on FTA cards………………………………………... 62 2.7 In vitro culture and cryopreservation of Leishmania isolates……………. 62 2.8 Use of haemcytometer for cell counting…………………………………. 63 Chapter Three 3.0 Morphological characterisation of the parasite…………………………... 64 3.1 Introduction………………………………………………………………. 64 3.2 Materials and Methods…………………………………………………… 67 3.2.1 Light microscope morphometry of Leishmania GH5………………….. 67 3.2.2 Electron microscopy of GH5…………………………………………… 68 3.2.2.1 Electron microscopy (EM) fixative…………………………………... 68 3.2.2.2 Promastigotes processing…………………………………………….. 68 3.2.2.3 Processing of promastigotes for SEM………………………………... 68 3.2.2.4 Processing of promastigotes for TEM………………………………... 70 3.3 Results……………………………………………………………………. 70 3.3.1 Culture………………………………………………………………….. 70 3.3.2 Morphometry of GH5 by light microscopy……………………………. 71 3.3.3 Electron Microscopy…………………………………………………… 73 3.3.3.1 Scanning electron morphology (SEM)……………………………….. 73 3.3.3.2 Transmission electron morphology (TEM)…………………………... 74 3.4 Discussions and Conclusions…………………………………………….. 76 Chapter Four 4.0 Molecular identification and analysis……………………………………. 79 4.1 Introduction………………………………………………………………. 79 8 4.2 Materials and Methods…………………………………………………… 84 4.2.1 Isolation/Extraction of DNA…………………………………………… 84 4.2.2 Molecular analysis……………………………………………………… 85 4.3 Results……………………………………………………………………. 89 4.3.2 Field investigation……………………………………………………… 89 4.3.3 Laboratory investigations………………………………………………. 92 4.1 Discussion………………………………………………………………... 96 Chapter Five 5.0 Vector Studies……………………………………………………………. 102 5.1 Introduction………………………………………………………………. 102 5.2 Exceptional vectors of emerging Leishmania species………………….... 105 5.3 Materials and Methods…………………………………………………… 107 5.4 Results……………………………………………………………………. 109 5.5 Discussion………………………………………………………………... 114 5.6 Conclusion………………………………………………………………... 122 Chapter Six 6.0 Rapid test diagnosis………………………………………………………. 123 6.1 Introduction………………………………………………………………. 123 6.2 Materials and Methods…………………………………………………… 129 6.3 Results……………………………………………………………………. 130 6.4 Discussion………………………………………………………………... 131 6.5 Conclusion………………………………………………………………... 135 Chapter Seven 7.0 Leishmania promastigotes susceptibility to cryptolepine………………... 136 7.1 Introduction………………………………………………………………. 136 7.2 Materials and Methods…………………………………………………… 142 9 7.2.1 Growth curves of isolates………………………………………………. 143 7.2.2 Amphotericin B susceptibility test……………………………………... 143 7.2.3 The alkaloid and its action on L. mexicana and Leishmania GH5…….. 143 7.2.4 Other factors influencing the effect of cryptolepine…………………… 144 7.3 Results……………………………………………………………………. 145 7.3.1 Susceptibility of L. mexicana promastigotes to Amphotericin B……… 146 7.3.2 Susceptibility of L. mexicana promastigotes to cryptolepine…………. 148 7.3.3 Other factors influencing the effect of cryptoplepine…………………. 150 7.3.4 Susceptibility of L. GH to cryptolepine………………………………... 153 7.4 Dose responses…………………………………………………………… 158 7.5 Discussion………………………………………………………………... 159 7.6 Conclusion……………………………………………………………….. 165 Chapter Eight 8.0 General Discussion……………………………………………………….. 167 8.1 Introduction………………………………………………………………. 167 8.2 Conclusions……………………………………………………………..... 168 8.3 Recommendations………………………………………………………... 174 8.4 Ongoing research priorities………………………………………………. 175 References………………………………………………………………….... 176 Appendix…………………………………………………………………….. 216 10 List of Figures Figure 1.1 Examples of various forms of cutaneous lesions.………………... 6 Figure 1.2 Multiple lesions indicative of DCL.……………………………… 7 Figure 1.3 Examples of PKDL showing numerous papular lesions ………… 11 Figure 1.4 The global outlook of leishmaniasis and Leishmania-HIV co- infection.…………………………………………………………………........ 12 Figure 1.5 The two major morphological forms of Leishmania parasite.…… 15 Figure 1.6 Overview of the life cycle of Leishmania in vector and mammalian hosts.…………………………………………………………….. 16 Figure 1.7 Different developmental forms ofL eishmania.………………….. 17 Figure 1.8 Location of parasite forms in the sand fly midgut…. ……………. 17 Figure 1.9 Movement of Leishmania parasites in the sandfly gut ………….. 18 Figure 1.10 Comparison of suprapylarian and peripylarian development of Leishmania.…………………………………………………………………... 19 Figure 1.11 Female sandfly taking a blood meal.…………………………… 28 Figure 1.12 Map showing new foci of leishmaniasis.……………………….. 33 Figure 1.13 Leishmaniasis mapping in Europe…. …………………………... 43 Figure 2.1 Map of Ho district showing endemic area.………………………. 57 Figure 3.1 The developmental order of the main promastigote forms.……… 66 Figure 3.2 Growth curve of the GH5 isol ate compared to that of L. mexicana.……………………………………………………………………... 70 Figure 3.3 Morphology of the Giemsa stained GH 5 from days 1 -7.………… 71 Figure 3.4 Daily average measurements of GH5 under the light microscope. . 72 Figure 3.5 Low power SEM view of GH5 promastigotes. …………………... 73 Figure 3.6 Rosettes and aggregates of GH5 promastigote s in SEM.………... 73 Figure 3.7 Montage of GH5 promas tigotes observed by SEM. ……………... 74 Figure 3.8 Various TEM sections thr ough GH5 promastigotes. …………….. 75 Figure 4.1 Some examples of lesions from participants in the study.……….. 90

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