Molecular Diagnosis of Invasive Fungal Disease Rebecca Louise Gorton UCL Infection and Immunity I, Rebecca Louise Gorton, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 1 Acknowledgments Firstly, I would like to express my sincere gratitude to my Ph.D. supervisors Prof. Christopher Kibbler and Prof. Tim McHugh for their continuous support throughout my Ph.D. Thank you for the many years of patience, motivation, and guidance. Your commitment to my Ph.D. was ever present throughout my research and the last months whilst writing my thesis. Without your support this would not have been possible. I would like to thank Dr Lewis White, who throughout my Ph.D. was a source of collaboration and inspiration as an expert in the field of molecular mycology. To Dr Emmanuel Wey, thank you for believing in me over last few years of collaborative service development. I am looking forward to many more exciting years of research. My sincere thanks also go to Shila Seaton who was my laboratory trainer in Mycology and inspired me every day to be a Mycologist. I would also like to thank my fellow laboratory colleagues from the NHS laboratory and UCL Infection and Immunity research group. Thank you for the stimulating discussions, for the sleepless nights working together before deadlines, and for all the fun we have had in the last eight years. There are too many of you to name but I hope all of you know how grateful I am to you. Last but not the least, I would like to thank my best friend and my family: to Janet and Philip for whom words will never be able to express just how grateful and lucky I am to have you as my parents. To my sisters, brother, sisters-in-law, brothers-in law, nieces and nephews, you have all allowed me to be away from home for so many years pursuing my career as a Mycologist. Without your continued love and support I could not have achieved so much. To Michelle Cairns who travelled along the Ph.D. pathway with me and was an inspiration and my close friend throughout, I’m sure we will look back on this and smile in years to come. Finally to my partner Michael, for the endless cups of tea whilst writing my chapters through to sitting with me in the final hours before my submission deadline willing me to complete this thesis. You have all been my inspiration and compass throughout. 2 Abstract Background Invasive fungal infections (IFI) are opportunistic infections caused by yeast or filamentous fungi, typically presenting in immunocompromised patients (haemato- oncology, intensive care, HIV, solid organ transplant settings). This research aims to comprehensively evaluate molecular diagnostics to address the current shortfall in IFI diagnosis and, where appropriate, embed molecular methods into routine clinical service. Methods Molecular methodologies, including PCR, MALDI-TOF MS and PNA-FISH, were evaluated on clinical sample cohorts from the Royal Free Hospital NHS Foundation Trust. Each method was critically appraised for: statistical performance, clinical utility and suitability for service adoption. Results MALDI-TOF MS improved yeast agar culture identification, demonstrating 97.4% (185/190) concordance with ITS rRNA sequencing, and time to identification was significantly reduced (p<0.01, 24 hrs. v’s 15 mins). Lower identification rates of 66% (33/50) were observed when applying MALDI-TOF MS directly to blood culture for yeast identification. In contrast PNA-FISH identified 98.5% (93/96, CI: 91.2, 98.9) of yeasts direct from blood culture within 30 minutes. Using PCP PCR a 60% (3/5) increase in the detection of PCP from BAL in non-HIV patients was demonstrated compared with GMS staining. Overall sensitivity was 100% (95% CI: 56.6, 100) and specificity was 97.9% (95% CI: 88.9, 99.6) for the diagnosis of PCP. Aspergillus PCR demonstrated a sensitivity of 100% (95% CI: 34.2, 100) and specificity of 93.8% (95% CI: 86.4, 97.3) for the diagnosis of IA but a low PPV of 28.6% (95% CI: 8.2, 64.1). Conclusions Molecular diagnostic assays can improve the diagnosis of IFI through improved accuracy of identification and increased detection of fungal pathogens from specimens. Results must be interpreted alongside clinical presentation, as false positivity occurs utilising highly sensitive molecular assays. Dual biomarker strategies may improve the performance of molecular diagnostics but the associated impact on healthcare economics must also be considered 3 Table of contents Acknowledgments ............................................................................................... 2 Abstract ............................................................................................................... 3 Table of contents ................................................................................................. 4 Abbreviations .................................................................................................... 11 Table of figures .................................................................................................. 13 1. Chapter 1 Introduction ............................................................................. 18 1.1 Global incidence and mortality of invasive fungal disease .................................... 18 1.2 Overview of Invasive fungal infections in the United Kingdom ............................. 20 1.3 Overview of Antifungal drugs ............................................................................. 21 1.4 Guidelines for the definition, diagnosis and management of invasive fungal disease 24 1.5 Overview of Immunity to fungal infection ........................................................... 25 1.6 Invasive fungal infections ................................................................................... 27 1.7 Invasive Candidiasis ............................................................................................ 27 1.7.1 Epidemiology of invasive candidiasis................................................................... 28 1.7.2 Clinical presentation of invasive Candidiasis ....................................................... 31 1.7.3 Laboratory diagnosis of Invasive Candidiasis ...................................................... 32 1.8 Pneumocystis pneumonia (PCP) .......................................................................... 39 1.8.1 Epidemiology of P. jirovecii infection .................................................................. 40 1.8.2 Clinical presentation of PCP ................................................................................. 40 1.8.3 Radiological diagnosis of PCP .............................................................................. 41 1.8.4 Microscopy for the detection of P. jirovecii ........................................................ 42 1.8.5 Serological diagnosis of PCP – (1-3)-β-D-glucan .................................................. 45 1.8.6 PCR for the diagnosis of PCP ................................................................................ 45 1.8.7 Prophylaxis and treatment of PCP ....................................................................... 46 1.9 Invasive mould infection ..................................................................................... 47 1.10 Invasive Aspergillosis .......................................................................................... 48 1.10.1 Epidemiology of Invasive Aspergillosis ................................................................ 48 1.10.2 Clinical presentations of Invasive Aspergillosis ................................................... 49 1.10.3 Laboratory diagnosis of Invasive aspergillosis ..................................................... 50 1.10.4 Histopathology of tissue sections ........................................................................ 51 1.10.5 Microscopy and culture of Aspergillus sp from clinical specimens ..................... 52 1.10.6 Serological diagnosis of Aspergillosis .................................................................. 54 1.10.7 PCR for the diagnosis of Invasive Aspergillosis .................................................... 55 1.10.8 Prophylaxis and treatment of invasive aspergillosis ........................................... 56 1.11 Non-Aspergillus invasive mould infection ............................................................ 57 1.11.1 Epidemiology of non-Aspergillus invasive mould infection................................. 57 1.11.2 Clinical presentation of Non-Aspergillus invasive mould infection ..................... 59 1.11.3 Laboratory diagnosis of Non-Aspergillus invasive mould infection .................... 61 1.11.4 Microscopy for the detection of non-Aspergillus IFI ........................................... 61 1.11.5 Fungal culture ...................................................................................................... 61 1.11.6 PCR for the detection and identification of non-Aspergillus fungi ...................... 62 4 1.11.7 Antifungal therapy and management of non-Aspergillus invasive fungal infection 63 1.12 Molecular diagnostic tool box ............................................................................. 64 1.12.1 Parameters used to determine the performance of a diagnostic test ............... 64 1.12.2 Polymerase chain reaction (PCR) ........................................................................ 66 1.12.3 Matrix assisted laser desorption ionisation time