A modified approach to recover and enumerate Ascaris ova in wastewater and sludge

Introduction The human roundworm, Ascaris lumbricoides, is the major soil-transmitted helminth (STH) of public health concern, with an estimated prevalence of approximately 1.2 billion people, especially in Southeast Asia, Latin America, and sub-Saharan Africa [1]. Poor sanitation, poor hygiene, and inadequate water supply are considered the major epidemiological factors of ascariasis. The reuse of wastewater and sludge as an alternative to freshwater in agriculture is partly responsible for widespread STH infections [2]. A low infective dose combined with extreme resistance to environmental stress and excretion of up to 200,000 ova per day in the faeces of infected hosts enhances the infectivity of Ascaris [3]. In many countries with good sanitation systems (e.g., Australia), ascariasis is not endemic in humans and Ascaris ova are not commonly detected in raw sewage [4, 5]. However, increased migration from Asia and Latin America, in addition to the growing rates of travel to developing countries, is likely to increase the incidence of ascariasis [6]. Therefore, the removal of Ascaris ova from wastewater in developed countries remains crucial to allow the safe use of sludge or recycled water in agriculture [7]. For the unrestricted use of wastewater in agriculture, the World Health Organisation (WHO) has formulated stringent guidelines and has recommended�1 viable Ascaris ova/L for treated wastewater (recycled water) and�1 viable Ascaris ova/g dissolved solids for sludge to minimise public health risk associated with Ascaris species [8]. As a result, the methods utilised for enhanced ova recovery plays a significant role in accurately enumerating their presence in wastewater and sludge samples. The United States Environmental Protection Agency (US EPA) has recommended the Tulane method for the detection and enumeration of STH ova in wastewater and sludge [9]. This method employs a series of steps, such as desorption, sedimentation, sieving, and flotation followed by enumeration using optical microscopy [10]. However, this method is time consuming because it takes approximately three days. Although various protocols have been developed to increase the recovery efficiency of Ascaris ova, until now there is lack of a universally acceptable method for the enumeration of STHs ova [11]. Therefore, the focus of this study was to develop a recovery method for Ascaris ova with minimal processing time and less damage to ova, without compromising the recovery efficiency and to compare the modified protocol with published methods.