Diagnostic methods for detection and characterisation of dimorphic fungi causing invasive disease in Africa: Development and evaluation TSIDISO GUGU MAPHANGA Diagnostic methods for detection and characterisation of dimorphic fungi causing invasive disease in Africa: Development and evaluation by TSIDISO GUGU MAPHANGA A thesis submitted in partial fulfilment of the requirements for the degree of PHILOSOPHIAE DOCTOR PhD (Medical Microbiology) in Department of Medical Microbiology Faculty of Health Sciences University of the Free State Bloemfontein, South Africa Promoter: A/Prof NP Govender March 2020 DECLARATION I, Tsidiso Gugu Maphanga, declare that the research thesis hereby submitted to the University of the Free State for the Doctoral Degree in Medical Microbiology and the work contained therein is my own original work and has not previously, in its entirety or in part, been submitted to any institution of higher education for degree purposes. I further declare that all sources cited are acknowledged by means of a list of references. Signed this day of 2020 Research is to see what everybody else has seen and to think what nobody else has thought Albert Szent-Györgi (US Biochemist) DEDICATION To my late brother, my mechanical engineer (Thabiso Benson Masilela): I will never forget all the plans we had, the motivations, love and laughter we shared. Life is never the same without you; I miss you daily. ACKNOWLEDGMENTS To God of Abraham, Isaac, Jacob, St Engenas and my God: Thank you for giving me strength, knowledge, wisdom and the ability to undertake this project. Your faithfulness reaches to the skies. A/Professor Nelesh P. Govender (supervisor): I would like to express my sincere gratitude for your patience, guidance, continuous support and useful comments received throughout this project. Your immense knowledge contributed greatly to my understanding of this project. Thank you for taking a chance on me and for always being optimistic even when things were not going according to plan. Professor Anwar A. Hoosen (mentor): no words can describe my gratitude. Thank you for seeing potential in me. I am grateful for the opportunities and support you have given me throughout my postgraduate studies. Thato Malele (husband): Thank you for your understanding, support and encouragement. Your prayers throughout this PhD have been amazing. You have been the pillar of my strength and you believed in me when I stopped believing in myself and for that I am forever grateful. Matlhatse Malele and Thato Jnr Malele (children): Thank you for keeping me sane; your presence in my life came just at the right time. Mavis Mashego and Happy Maphanga (parents); Mlungisi, Phumla, Sabelo, Lesego and Tumelo (siblings): Thank you for supporting me spiritually throughout my academic years. Your love and words of encouragement kept me going. Ruth Mpembe (laboratory manager): Thank you for your support and words of encouragement. Samaria Mafuma, Iqra Barakzai, Dr Adeola Rotimi, Dr Nontutuko Maningi, and Dr Nomcebo Sengwayo (friends): I am grateful for your patience, support and words of encouragement, but most of all for the love you showed me when I needed you most. Serisha Naicker, Mabatho Mhlanga, Mbali Dube, Ivy Rukasha and Sydney Mokgokotleng (colleagues): Thank you for making my workplace more constructive to work in. Your amazing support and words of encouragement received during this period were much appreciated. Thokozile Gloria Zulu (my mycologist): Thank you for sharing your knowledge. The fun we had in the laboratory learning about fungi was amazing. I am also thankful for the words of encouragement, your support and always making sure that I was well taken care of. I am in debt to you; may God truly bless you. Dr Monica Birkhead: Thank you for your tremendous support, your insightful comments and encouragement. You made me believe that it is possible. TABLE OF CONTENTS Page LIST OF FIGURES vii LIST OF TABLES viii LIST OF UNITS AND ABBREVIATIONS ix LIST OF PUBLICATIONS xii NATIONAL CONFERENCE PRESENTATIONS xiv ABSTRACT xvi CHAPTER 1: 1.1 Introduction 1 1.2 Thesis structure 4 References 5 CHAPTER 2: LITERATURE REVIEW 2.1 Introduction 7 2.2 Classification of dimorphic fungal pathogens 8 2.2.1 Phylogenetic relationship of dimorphic fungal pathogens 8 2.3 General characteristics of dimorphic fungi 11 2.3.1 Dimorphism 14 2.3.2 Virulence factors of dimorphic fungi 14 2.4 Clinical manifestations of dimorphic fungal infections 15 2.4.1 Emergomycosis 16 2.4.2 Blastomycosis 17 2.4.3 Histoplasmosis 19 2.4.4 Sporotrichosis 20 2.5 Prevention of acquisition of dimorphic fungal pathogens 21 2.5.1 Treatment of dimorphic fungal infections 22 2.5.2 Antifungal susceptability testing of dimorphic fungal isolates 23 2.6 Eco-epidemiology of dimorphic fungal infections 25 2.7 Diagnostic methods for the identification of dimorphic fungi 31 2.7.1 Histopathology method for the identification of dimorphic fungi 31 i Page 2.7.2 Culture-based method for the identification of dimorphic fungi 32 2.7.3 Antigen detection method for the identification of dimorphic fungi 33 2.8 Molecular methods for the detection and characterisation of dimorphic 35 fungi 2.8.1 Conventional and quantitative PCR for the detection of dimorphic fungi 35 2.8.2 Characterisation of dimorphic fungi using multilocus sequence typing 36 2.8.3 Whole genome sequencing and single nucleotide polymorphism (SNP) 38 analysis of dimorphic fungi 2.9 Summary 41 References 44 CHAPTER 3: In vitro antifungal susceptibity of yeast and mould phases of 65 isolates of dimorphic fungal pathogen Emergomyces africanus (Formerly Emmonsia sp.) from HIV-infected South African patients 3.1 ABSTRACT 65 3.2 INTRODUCTION 66 3.3 MATERIALS AND METHODS 68 3.3.1 Isolates and case definition 68 3.3.2 Identification to the species level 69 3.3.3 Antifungal susceptibility testing 69 3.3.4 Statistical methods 70 3.3.5 Ethics approval 70 3.4 RESULTS 70 3.4.1 Cases and isolates 70 3.4.2 Antifungal susceptibility testing of Emergomyces isolates 71 3.4.3 Minimum inhibitory concentration distribution and patient outcome 74 3.5 DISCUSSION 76 3.5.1 Study limitations 78 3.6 CONCLUSION 78 ii Page 3.7 ACKNOWLEDGMENTS 78 3.8 FUNDING 78 3.9 CONFLICTS OF INTEREST 78 3.10 AUTHORS’ CONTRIBUTIONS 78 REFERENCES 80 CHAPTER 4: A diagnostic accuracy case-control study to evaluate a 83 Histoplasma capsulatum urine antigen enzyme immunoassay among patients with suspected histoplasmosis 4.1 ABSTRACT 84 4.2 INTRODUCTION 85 4.3 MATERIALS AND METHODS 86 4.3.1 Participants, specimens and case definitions 86 4.3.2 Detection of Histoplasma antigen 87 4.3.3 Species confirmation 87 4.3.4 Statistical methods 88 4.3.5 Ethics approval 88 4.4 RESULTS 88 4.4.1 Cases with suspected histoplasmosis 88 4.4.2 Clinical details of 171 particitipants with or without invasive fungal 89 disease 4.4.3 Detection of Histoplasma capsulatum antigen from urine 89 4.5 DISCUSSION 90 4.5.1 Study limitations 93 4.6 CONCLUSION 94 4.7 ACKNOWLEDGMENTS 94 4.8 FUNDING 94 4.9 CONFLICTS OF INTEREST 94 4.10 AUTHORS’ CONTRIBUTIONS 94 REFERENCES 95 iii Page CHAPTER 5: Human blastomycosis in South Africa caused by Blastomyces 105 percursus and Blastomyces emzantsi sp. nov., 1967-2014 5.1 ABSTRACT 106 5.2 INTRODUCTION 106 5.3 MATERIALS AND METHODS 107 5.3.1 Case detection and isolate archive 107 5.3.2 Morphological description and electron 108 5.3.3 Antifungal susceptibility testing 108 5.3.4 DNA extraction, PCR amplification and sequencing 109 5.3.5 Multilocus sequence typing phylogenetic analysis 109 5.3.6 Whole genome sequencing and de novo assembly 109 5.3.7 Phylogenetic analysis using whole-genome single nucleotide 109 polymorphisms 5.3.8 Statistical methods 110 5.3.9 Ethics approval 110 5.4 RESULTS 110 5.4.1 Isolates and cases 110 5.4.2 Mycological examination of the isolates 110 5.4.3 Antifungal susceptibility testing of isolates 111 5.4.4 Multilocus and whole genome phylogenetics analyses 111 5.4.5 Clinical presentation and treatment of 10 cases 112 5.4.5.1 Group one isolates resembling Blastomyces percursus 112 5.4.5.2 Group two isolates resembling Blastomyces emzantsi 112 5.4.6 Genome assembly sizes 112 5.4.7 Nucleotide diversity 112 5.4.8 Mating identification from whole genome sequencing 113 5.4.9 BAD-1 gene identification from whole genome sequencing 113 5.4.10 Taxonomy 113 5.5 DISCUSSION 114 5.5.1 Study limitations 116 5.6 CONCLUSION 116 iv Page 5.7 ACKNOWLEDGMENTS 117 5.8 FUNDING 117 5.9 CONFLICTS OF INTEREST 117 5.10 AUTHORS’ CONTRIBUTIONS 117 REFERENCES 118 CHAPTER 6: Molecular identification of dimorphic fungi causing systemic 134 mycoses among South African patients 6.1 ABSTRACT 135 6.2 INTRODUCTION 136 6.3 MATERIALS AND METHODS 137 6.3.1 Fungal isolates and case definition 137 6.3.2 Molecular identification from cultured isolates 138 6.3.3 Genus confirmation by phenotypic methods 138 6.3.4 Statistical methods 139 6.3.5 Ethics approval 139 6.4 RESULTS 139 6.4.1 Isolates and cases 139 6.4.2 Identification of dimorphic fungi by phenotypic methods at the 140 diagnostic laboratories 6.4.3 Identification of dimorphic fungi by phenotypic methods at the 140 reference laboratory 6.4.4 Identification of dimorphic fungi by DNA- sequencing methods at the 141 reference laboratory 6.4.5 Clinical presentation of the 156 cases with systemic mycosis 141 6.5 DISCUSSION 143 6.5.1 Study limitations 146 6.6 CONCLUSION 147 6.7 ACKNOWLEDGMENTS 147 6.8 FUNDING 147 6.9 CONFLICTS OF INTEREST 147 6.10 AUTHORS’ CONTRIBUTIONS 147 v Page REFERENCES 148 CHAPTER 7: CONCLUSION 157 7.1 Concluding remarks 157 7.2 Future research 160 REFERENCES 163 Supplementary Appendix: Human blastomycosis in South Africa caused by 165 Blastomyces percursus and Blastomyces emzantsi sp.