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A comprehensive investigation of existing sanitation helminth enumeration methods with the aim of producing an international standard.

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TeHelminth testing in faecal sludge should be consistent so data are comparable. New faecal sludge treatments for on-site toilet technologies are constantly being developed in order for municipalities in developing countries to supply dignified alternatives to sewered systems that waste large amounts of potable water and require pumping to wastewater treatment works for centralised treatment. In order to ensure that these new, onsite toilet technologies adequately sanitise the faecal matter, helminth eggs are spiked into these systems to test inactivation according to the ISO-30500 standard for non-sewered sanitation systems (NSSS). A sensitive, standard helminth isolation and enumeration method, accredited to the ISO-17025 international standard for testing and calibration laboratories, is therefore required for application in laboratories globally. Internationally, laboratories and groups have used variations of the standard United States Environmental Protection Agency (USEPA) Method (2003), the Mexican Standard for Wastewater Analysis (2012), the Bailenger Method (1996) and the Pollution Research Group (PRG) Helminth Method (2017) previously used by the Water, Sanitation and Hygiene Research and Development Centre (WRDC) for helminth testing, and formed the foundation of this study. Conventional helminth methods can be broken down into five steps: washing and sedimentation of samples to separate eggs from larger particles, flotation using density gradients to separate eggs from heavier particles, centrifugation after both washing and flotation, extraction, that involves the use of a buffer and solvent combination to further separate organic material from eggs, and microscopic analysis. Some methods also include incubation that allows for egg-viability assessment. Every reagent used in these helminth methods was tested on Ascaris suum eggs for varying time intervals; ammonium bicarbonate and 7X® (a brand of ionic surfactant) performed best in terms of egg development and viability. Washing samples under pressure and no pressure were compared and the former produced the best egg recovery. Different flotation solutions were tested at different specific gravities, and zinc sulphate at specific gravity of 1.3 recovered the most eggs. Centrifugation speeds and times were tested after the washing and flotation steps, and 3000 rpm for 10 minutes and 2000 rpm for 15 minutes produced optimal egg recovery, respectively. Different extraction combinations were tested, and it was discovered that eggs were lost in this step. It was therefore recommended that extraction be removed from the method. Different wash solutions were then tested against various sample types to determine which resulted in the highest percentage egg recovery and which solutions facilitated easier microscopic analysis. Based on data from each experiment, a final SOP was produced for the new WRDC Helminth Method, that accommodates different sample types and egg viability assessment post method.


Doctoral Degree. University of KwaZulu-Natal, Durban.