Date of Award
Doctor of Philosophy
Prof. Roy Sleator
Dr Brigid Lucey
Cryptosporidium remains an underestimated, yet highly prevalent, parasitic agent of gastrointestinal illness worldwide. In clinical settings, diagnosis has previously relied on microscopic techniques. However, enteric parasite targeting molecular tools, such as real-time PCR panels, have superseded microscopy in recent years. This body of work examines the nature, implications and opportunities of this paradigm shift. This thesis is divided into seven chapters that consist of a literature review chapter, five experimental chapters focusing on the molecular diagnosis and epidemiological analysis of Cryptosporidium spp., and a final summary chapter. Chapter I provides the contextual narrative and supportive reasoning behind the research undertaken in this thesis through discussion of the published literature. A clinical validation study, introducing a real-time PCR based method for the clinical diagnosis of cryptosporidiosis to the Medical Microbiology Department of Cork University Hospital, and an examination of the impact of reducing routine enteric microscopy on detection rates of other enteric parasites provided the initial impetus behind this research. These studies are outlined and discussed in Chapter II. Chapter III details the longitudinal epidemiological study that was conducted in collaboration with Cork University Hospital on a bank of clinical Cryptosporidium samples amassed through routine clinical testing. Routine diagnostic testing in this medical microbiology laboratory was limited to genus level identification. This study identified the species implicated in clinical infection among the amassed isolates through fluorescent probed-based real-time PCR analysis of the 18S rRNA gene. Further epidemiological and phylogenetic analyses were conducted on the amassed clinical Cryptosporidium sample bank to identify isolates to the subspecies/subtype level. This was achieved thorough sequencing of the 60-kDa glycoprotein gene. This analysis resulted in the discovery of Cryptosporidium parvum and Cryptosporidium 8 hominis gp60-subtypes hitherto not detected in Ireland. This research is detailed in Chapter IV. The diversification of the Cryptosporidium population in Ireland and the general heterogeneity of Cryptosporidium populations reported globally provided the basis for the research conducted in Chapter V. This chapter describes the development of a novel methodology to differentiate between C. parvum gp60-subtypes, without the need for DNA sequencing, through the real-time PCR application, high resolution melting analysis. Chapter VI describes the application of an analogous subtyping method to that developed in the preceding chapter to differentiate between C. hominis gp60-subtypes. Chapter VII provides a summary of the research described herein and also details future avenues of research for the advancement of the developed HRM analysis-based Cryptosporidium spp. subtyping methods.
O'Leary, Jennifer K., "Novel Molecular Approaches to Diagnostic and Epidemiological Analyses of Cryptosporidiumspp.Implicated in Clinical Infection" (2022). Theses [online].
Available at: https://sword.cit.ie/allthe/42
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