Doctor of Philosophy
School of Civil, Mining and Environmental Engineering
Saaed, Frhat M. A., Measurement of Cryptosporidium and Giardia concentrations in water using conventional and molecular methods, Doctor of Philosophy thesis, School of Civil, Mining and Environmental Engineering, University of Wollongong, 2011. http://ro.uow.edu.au/theses/3648
The concentrations of both Cryptosporidium oocysts and Giardia cysts in environmental water are low. The ability to accurately detect, count, and manipulate small numbers (i.e. 1 to 100) of these oocysts and cysts in surface water with different levels of water quality ranging from low to high turbidity is considered essential for the prevention of gastroenteritis associated with Cryptosporidium and Giardia. The ability to accurately count and manipulate small numbers of both organisms was systematically demonstrated. In addition, the reported data on the statistical distribution of organisms found in comparison to the theoretical Poisson model have shown that the direct cause of the observations of negative (zero) findings from application of current monitoring of 10 L surface water sources of public water supply samples using USEPA Method 1623 analysis is that the limit of detection, dependent only on the sample volume and recovery efficiency of the analysis in the specific water, is above the ambient concentration. Water samples were collected from four coastal streams draining upland forested and agricultural land in SE New South Wales in typical seasonal periods. Sample volumes were sufficient to enable finding Cryptosporidium and Giardia in the majority of samples. The oocyst concentration ranged from less than 0.07 to about 5.2 oocysts/L, while the cysts concentration ranged from less than 0.04 to 7.7 cysts/L. The detection limit of the immunomagnetic separation (IMS) method-based on method 1623 in surface water samples covering a range of ambient water quality ranging up to turbidity between 0.76 to 35 NTU was defined. The detection limit varied depending on the type of the water sample and ranged from about 1.0 oocysts/L to as low as 0.03 oocysts/L, while the cysts limit of detection ranged from 0.02 to 0.24 cysts/L. The average recovery rate of the IMS method 1623 was resulted in typical recovery efficiencies of (25%) for oocysts and 58% cysts. The results of IMS-based method have shown a differential response in a variety of water types and it yielded highly variable results depending on the water type.
The testing and evaluation of previously described loop-mediated isothermal amplification (LAMP) assays and exploration of their quantitative characteristics were pursued using Cryptosporidium control DNA and control DNA samples of Giardia. The results showed that the LAMP assay has high sensitivity and ability to amplify a single copy of the gene target resulting in a low detection limit for the assay.
The oocyst and cyst quantification LAMP assays explored here represent a promising alternative to immunofluorescence assay (IFA) and microscopy method for the detection of both parasites in water samples. The LAMP assay was able to detect DNA extracted from a single oocyst or cyst. With further development, LAMP should be a useful alternative for quantifying total levels of both organisms and makes an important direct contribution at a relatively low cost to test for the presence and concentration of these organisms in water in comparison with the standard methods currently in use. Use of suitable primers also offers potential for species discrimination using the LAMP process.