Detection of viable hookworm ova from wastewater and sludge

Abstract

Infectious helminths are a worldwide major public health problem. In terms of morbidity, approximately 3 × 109 people are infected by soil-transmitted helminths (STHs) influencing rates of malnutrition and failure to thrive. All of this increases the global disease burden (GDB) by up to 5.9 × 106 Daily Adjusted Life Years (DALYs). Helminth infections are more often seen as a major public health issue for developing countries however, concerns have been expressed in many of the developed nations because of the increased use of treated wastewater and sludge with no clear way of knowing the infection potential that can be attributed from such water. Guidelines have been established to minimise the potential public health risk associated with wastewater and sludge reuse. For example, the WHO guideline specified ≤ 1 ova (Ascaris lumbricoides) per L liquid (wastewater) or 4 g dry solid (sludge) for unrestricted use. Various wastewater treatment processes have been recommended to remove the helminths ova from wastewater depending upon its reuse. However, detection methods used to quantify viable helminths ova from wastewater has limitations. Therefore there is always a potential public health risk associated with reuse of wastewater and sludge. In this research, a real-time PCR method was developed. The new PCR method is rapid and specific. The method was found to be able to detect less than one ovum in one litre treated wastewater and approximately four ova in one litre raw wastewater and ~ 4 g sludge. The real-time PCR method was modified to a quantitative PCR (qPCR) method and further used to quantify hookworm ova from wastewater and sludge. The qPCR had estimated an average of 1.1, 8.6 and 67.3 ova for treated wastewater that was seeded with 1, 10 and 100 ova, respectively. The gene copy numbers obtained for 1, 10 and 100 ova by qPCR varied significantly (P < 0.05) within the tested samples indicating that absolute quantification of ova may not be accurate. Despite the difficulty quantifying accurate numbers of hookworm ova, the lower limit of quantification (LLOQ) of the qPCR method was 30 gene copies. This was a lot less than the gene copies produced by one ovum. Therefore, the qPCR method has potential to use for complying with wastewater guidelines. Although the overall aim of this research was to develop a sensitive and specific method for quantitative detection of viable hookworm ova from wastewater, the importance of recovering the ova from wastewater and sludge samples for accurate detection was identified. While determining appropriate recovery rates was not an aim of this research, a suitable method that could be used to standardise research outcomes for further study was established. Therefore, ova recovery rate by different rapid methods was evaluated for further experiments. The result indicated that the ova recovery rate was higher for the treated wastewater (0.2 - 50%) than the raw wastewater (0.3 - 35%) and sludge (0.02 - 4.7%) samples. A significant difference (P < 0.05) was observed between the methods used to recover the ova from wastewater matrices. Therefore, using this newly developed detection method more research is needed in another study to improve the ova recovery rates from wastewater and sludge samples. It is well known that the qPCR detection method cannot distinguish between viable and non viable microorganisms (cells, oocysts, and ova). Since only viable ova are capable of becoming infectious larvae and consequently causing infections in humans, it is important for health risk assessments to know what fraction of the PCR amplified ova are viable. Therefore, the developed qPCR method was combined with Propidium Monoazide (PMA) to develop a selective detection method for detecting viable hookworm ova from wastewater. Results confirmed that the PMA-qPCR method did not detect hookworm ova in samples that had been treated to kill. The performance of viability quantification of the newly developed PMA-qPCR method was then assessed against currently available (culture based and vital stain) methods. The percentage of in vitro viability assessed by PMA-qPCR was 19% lower than vital stain and 38% higher than culture based methods. In general, the outcome of this research is an improved detection method which is sensitive and more specific than current method for detection of hookworm ova from wastewater. The method is rapid, cheap and does not require skilled personnel like microscopic methods. Therefore, it will be suitable for health regulators as well as wastewater utilities for quality control and risk assessment purposes. However, studies are required to determine the gene copy numbers in different cell stages of hookworm ova, in order to improve the accuracy of quantification using qPCR method.