Development of concentration and detection techniques for Cryptosporidium and Giardia and the significance of these protists in KwaZulu-Natal.

Abstract

Cryptosporidium and Giardia are waterborne parasitic protozoons that have been associated with some diarrhoeal illness in most parts of the world. They are considered of major importance for drinking water safety as waterborne outbreaks still occur regularly in both developed and developing countries, sometimes with fatal consequences. To determine their incidence in the KwaZulu-Natal population, an epidemiological review of pathology laboratory data was compiled. Both protists were found to be endemic although their occurrence did not appear to correlate with climatic factors such as rainfall, season or year, possibly indicating that other factors such as personal hygiene, potable water supply, sanitation and education probably have a more significant impact here rather than waterborne transmission. The results, however, may not be representative of the entire population as detection techniques are not standardised and data were only collected from laboratories willing to supply in the Durban and Pietermaritzburg areas which represent under 10% of laboratories in KwaZulu-Natal that test for Cryptosporidium and Giardia. These laboratories, however, perform most of the testing from throughout KwaZulu-Natal and are situated in the metropolitan areas. In addition, poor recording of patient details made conciling of data difficult. Evaluation of a calcium carbonate flocculation, membrane filtration and membrane dissolution technique for concentrating Cryptosporidium oocysts and Giardia cysts from water is necessary to accurately quantify (oo)cysts in water. Methods currently used result in varying recoveries and the best method had to be identified and enhanced for this study. Greater (oo)cyst losses occurred as turbidity increased irrespective of the method used. The calcium carbonate flocculation method proved to have the best recovery for all water types and is recommended for use in the regular routine monitoring of smaller volumes of water. Pre-filtration prior to flocculation had the potential to make microscopy easier although losses of (oo)cysts still occurred. Sucrose flotation following flocculation reduced recovery whilst pH adjustment to 6 ± 0.5 following flocculation improved recovery and is recommended with turbid water samples. A cheap and simple detection method using the slide immunoenzymatic assay (SIA) which is based on the principles of enzyme linked immunosorbent assay (ELISA), was adapted to detect Cryptosporidium and Giardia in potable and turbid water concentrates. The results were reproducible and sensitivity was improved using a spectrophotometer. A multi-phase SIA system, using reagents in liquid and dry ready-to-use forms, was investigated and the potential exists for its further development. Unfortunately this method does not indicate the viability status of (oo)cysts therefore a novel method to do so was developed using fluorescein diacetate (FDA) and tetramethyl red (TMR) labelled anti- Giardia monoclonal antibodies. This combination stained viable cysts green internally with a red wall while non-viable cysts stained red only. While the use of FDA overestimates viability, any error would err in favour of safety, and could be complemented with fluorescein isothiocyanate / propidium iodide for confirmation of viability status. As Cryptosporidium and Giardia were found to be present in the KwaZulu-Natal population, monitoring of water bodies, water supplies, wastewaters and sludges, using the enhanced flocculation procedure and immunofluorescence assay, was undertaken to determine their source. Oocysts and cysts were detected in dam, river and raw waters, treated effluent and sludge samples. No oocysts or cysts were detected in the treated water samples although this may be due to the inability of the method used to detect low numbers of (oo)cysts. This research confirmed the occurrence of Cryptosporidium and Giardia in the KwaZulu- Natal population and water matrices. The optimum concentration method for use with water samples was established and further enhanced for use with turbid waters while a simpler and cheaper means of detecting (oo)cysts and a novel viability-detection stain for cysts were developed.