Cryptosporidium is a common water bourn protozoan pathogen. Its presence in active form in the water supply can pose a significant risk to community health. By quantifying Cryptosporidium oocyst inactivation instead of just detecting its removal, a more complete assessment of the Cryptosporidium risk can be obtained. Detection of inactivation can reduce treatment costs for recycled water by making further steps, intended to fully
remove oocysts, redundant. This project was about the optimisation of an assay to allow accurate quantification of oocyst inactivity.
The project examined the optimisation of a cell culture infectivity assay known as the “Focus Detection Method”. The aim was to develop a measure of Cryptosporidium infectivity that can quantify the inactivation of Cryptosporidium across the wastewater treatment train.
This assay works by creating the conditions where any remaining active Cryptosporidium oocyte goes to the next stage of its reproduction cycle in which it produces four sporozoites. A monolayer of host detection cells are
infected by the sporozoites thus creating a focus of in vitro infection that can be detected, revealing the presence in the sample of the active Cryptosporidium oocyte.
Adjustments to the assay included:
- A centrifuge step to more securely attach active oocytes to the detection monolayer;
- A reduction in pre-treatment temperature to ensure oocytes reached the monolayer before the release of sporozoites, thus ensuring each active oocyte produces just one infection focus in the detection monolayer;
- Addition of a wash step to remove oocytes from the detection layer was found to improve the in vitro cryptosporidium growth leading to improved sensitivity; and
- Optimisation of the concentration of bovine bile required to induce active oocytes to release sprozoites. This promotes in vitro infectivity and improves the tests sensitivity.
For this optimised assay, the limit of detection was found to be approximately 2-3 active oocysts per sample. The new assay is capable of in vitro detection of both C. hominis and C. parvum oocysts, the two most medically important cryptosporidium species that infect water supplies.
The Cryptosporidium cell culture infectivity assay is greatly improved, with a four-fold increase in sensitivity. This assay allows accurate quantification of oocysts inactivation in various stages of the wastewater treatment train. By quantifying oocyst inactivation instead of just their removal, a more complete assessment of Cryptosporidium oocyst risk is obtained, leading to cost reductions in provision of “fit for purpose” recycled water by eliminating the need for further, unnecessary treatment steps.