Microfluidics for the detection of Cryptosporidium
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This thesis details the development of microfluidics for the label-free sorting and/ or identification of waterborne pathogens which are commonly detected in contaminated drinking-water supplies using the United States Environmental Protection Agency method 1623.1 (USEPA 1623.1). This method recovers and detects pathogens of the Cryptosporidium and Giardia species, which can cause human gastroenteritis upon ingestion. USEPA 1623.1 is employed universally in developed regions (e.g., Europe, North America, Australia, New Zealand). Specifically, this thesis describes microfluidic systems that were developed with the objective of rapidly discriminating viable (i.e., intact and apparently infectious), humanpathogenic Cryptosporidium oocysts from non-viable, human-pathogenic oocysts and/ or species which are considered non-hazardous to human health. Such a system would reduce the overall detection time and allow a more accurate assessment of the risk posed to human health. A microfluidic setup incorporating dielectrophoresis was designed and employed for the viability-based sorting and enumeration of a human-pathogenic species of Cryptosporidium. This device enabled the sorting of untreated (live) and heat-inactivated (non-viable) sub-populations of the human pathogenic Cryptosporidium parvum with over 80% efficiency. Existing Microfluidic Impedance Cytometry (MIC) and Microfluidic-enabled Force Spectroscopy (MeFS) technologies were adapted for the enumeration, detection and viability determination of human-pathogenic Cryptosporidium oocysts, plus the discrimination of Cryptosporidium species which pose a major risk to human health from those which pose little to no risk. Using MIC, it was possible to discriminate untreated and heat-inactivated C. parvum with over 90% certainty. Furthermore, populations of C. parvum, Cryptosporidium muris (low-risk, human pathogen) and Giardia lamblia (also recovered using USEPA 1623.1) were discriminated from one another with over 90% certainty. Using MeFS, it was possible to differentiate temperature-inactivated (either by freeze- or heat-treatment) C. parvum from live C. parvum with a minimum of 78% efficiency. Finally, the high-risk, human pathogenic C. parvum was discriminated from C. muris with over 85% efficiency. Upon further validation, i.e., the analysis of other Cryptosporidium species and of oocysts which have been inactivated by other means (e.g., ozonation, ultraviolet radiaton), it is hoped that water utilities will employ such method(s) to more accurately characterise the human risk associated with contaminated supplies.