A chemiluminescent immunofocus assay (CIFA) for non-microscopic enumeration of Cryptosporidium parvum infectivity in cell culture.

ABSTRACT

As Cryptosporidium parvum continues to cause waterborne disease, despite extensive efforts by drinking water suppliers and regulators, it is important to have reliable and convenient methods for detection of this pathogen in wastewater discharges, environmental source waters and finished drinking water supplies. In order to better understand the health risks of this organism, it is necessary that detection methods be able to distinguish between infectious and non-infectious Cryptosporidium oocysts in these environmental samples. Cryptosporidium infectivity assay systems based on infections in mice and on in vitro infections in continuous mammalian cell lines are available. Currently, these methods are impractical for routine analysis of water samples because they are tedious, lengthy and costly. These methods rely on careful microscopic examination or further analysis by PCR and then characterisation of the amplified DNA. Practical and affordable non-microscopic methods to determine Cryptosporidium infectivity are much needed for environmental analysis. A cell culture infectivity detection system was developed for infectious Cryptosporidium oocysts that does not rely on microscopic examination of samples to score results, is applicable to a variety of samples and has the potential to be used for routine water monitoring and other environmental or biomedical analysis. Using a chemiluminescent immunoassay, the discrete foci of developmental stages of Cryptosporidium in cell cultures are clearly visible as discrete objects in an image of the entire cell culture layer in a dish or on a slide. These objects are directly countable with the unaided eye and their identity can be further confirmed or verified by microscopic examination.