Development and evaluation of robust detection methods for soil transmitted helminths of public health importance in wastewater

Ascaris lumbricoides, a major soil-transmitted helminth infecting over 1.2 billion people worldwide, is most refractory to treatment processes and its presence and/or inactivation in treated material can serve as an indicator of the efficiency of the disinfection process. Due to its high infectivity, risk to children, and resistance to adverse conditions such as high temperatures and alkaline pH, the Ascaris genus are parasites of great significance for evaluation of the sanitization of wastewater and sewage sludge. The reliable quantitative detection of viable ova is therefore essential for both accurate assessment of risk based target values and for the validation of the performance of sanitation systems. Conventional methods of helminth ova enumeration such as microscopy are time-consuming and tedious. The development of polymerase chain reaction (PCR)-based techniques have shown some success in overcoming a number of the challenges associated with conventional techniques, but these techniques also have shortfalls. The overarching aim of this thesis was to develop methods that assure high accuracy, speed and precision in the detection and determination of the viability of Ascaris ova in wastewater. In addition, the importance of accurately estimating the concentration of ova from wastewater and sludge samples was identified. Therefore, a new method to enumerate and recover Ascaris suum ova in wastewater and sludge was developed. The effectiveness of the modified method was compared with the Tulane and double flotation methods. The modified method resulted in enhanced ova recovery from wastewater (> 50%) and sludge (> 60%) with minimal processing time (≤ 2 hours). A significant increase in recovery (P value < 0.0001, paired T-Test) was observed when used to recover ova from wastewater and sludge. PCR-based techniques, especially quantitative PCR can be used to detect Ascaris ova in wastewater matrices with improved sensitivity and precision. However PCR/qPCR methods are unable to differentiate between viable and non-viable ova and might lead to overestimation of infection intensity. To overcome such limitations, a method was developed using DNA intercalating dye propidium monoazide (PMA) with qPCR for the selective detection of viable Ascaris ova from wastewater. The validated PMA-qPCR method was subsequently compared with existing culture-based and BacLight Live/Dead staining methods for viability determination. The percentage of viability determined were; culture-based (82%), BacLight Live/Dead staining (87%) and PMA-qPCR (85%), respectively. No significant statistical difference (P > 0.005, Fisher Exact Test) among the three methods was determined; however PMA-qPCR based viability determination is preferable due to its speed and accuracy. Thus PMA-qPCR could be a useful tool in the screening of Ascaris infection after mass anthelminthics administration and water sanitation and hygiene implemented countries.    Due to the complexity of the assays and requirement for specialised and sophisticated devices, PCR-based techniques can not readily be adapted to be a point-of-care detection assay or used in resource-limited settings where the disease is mostly endemic. To meet the need for rapid on-site assay in resource-limited settings, recombinase polymerase amplification (RPA) assay coupled with eco-friendly lateral flow (LF) strips was developed and validated for the detection of Ascaris ova in wastewater. The Ascaris RPA-LF assay was able to detect Ascaris in less than 30 minutes with an optimal temperature at 37 ¿C and was more sensitive than PCR-based approaches, with detection at DNA concentrations as low as 2 femtograms. Furthermore, ova from two different helminth genera, Ascaris suum and Trichuris suis were able to be detected as a multiplex RPA-LF assay which could significantly reduce the time and cost of helminth identification. The RPA-LF assay was a sensitive, specific, user-friendly and cost-effective technique for the rapid detection of helminth ova with significant potential as an on-site molecular detection tool. One of the main hindrances for the effective use of molecular methods in the detection of STH ova is the extraction of genomic DNA of good quantity and quality which is due to the presence of the tough outer shell of helminth ova. Commercial DNA extraction kits are generally optimised for the extraction of bacterial genomes; however the ova shell is much tougher than the cell walls of bacteria resulting in a lower DNA yield which affects the sensitivity of subsequent molecular assays. Hence, a simple colorimetry-based assay using the surface property of Ascaris ova for in-situ biosynthesis of gold nanoparticles via reduction of tetrachloroauric acid was developed as an alternative to molecular techniques. Visualisation of a colour change from light yellow to ruby red indicated the synthesis of gold nanoparticles on the surface of Ascaris ova; however no such colour change was observed in Trichuris ova. Scanning electron microscopy (SEM) revealed the outer surface of A, suum ova covered with nanoparticles while elemental analysis using energy dispersive X-ray spectroscopy (EDX) confirmed that the presence of 91 w/w % gold in the ova surface.  Although preliminary experiments were successful in terms of differentiating helminth ova, further validations with wastewater samples are required. The aforementioned studies to quantitatively detect Ascaris ova in wastewater represent significant innovations. Although all methods require further assessment, the research carried out in this thesis has led to significant findings in the diagnostic capabilities for STH, especially Ascaris ova, the neglected pathogen. Consequently, this thesis will support the water companies in Australia via accurate detection and screening of Ascaris in wastewater matrices thus allowing safe reuse of wastewater