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Precision and accuracy of an assay for detecting Ascaris eggs in various biosolid matrices
Abstract
This paper presents quality assurance data and quality control data on the recovery of Ascaris suum eggs from various biosolid matrices: acid-treated, alkaline-treated, amended-soil blended, and lagoon stored biosolids. Over a period of years, the same procedure, the "Tulane Method," was performed on different matrices, and in this work, the data collected on the recovery of the eggs from the different matrices is examined and compared. The egg recoveries are discussed in terms of precision (the comparison of the recovery of eggs from a sample processed in duplicate) and in terms of accuracy (the percentage of eggs recovered from a sample to which eggs were added at the beginning of the extraction procedure). This form of quality analysis/control is typically called the "Split/Spike" method. This method of biosolid processing for helminth egg recovery had an overall accuracy of about 60% or greater and a percent variation from the mean density (an indirect method of assessing precision) of only 3-35%.
... The most widely used method of assessing the viability is incubation. Solutions of sulfuric acid (24,25) and formalin (26) are used to incubate the isolated eggs at incubation temperatures ranging between 22°C and 26°C and duration of the incubation period between 21 and 30 days (26). The solution of sulfuric acid (0.1N) was reported to give the best result, between 75-80% (22) and 83-92% (27) viability, followed by formalin solution (75-80%) (26). ...
... The most widely used method of assessing the viability is incubation. Solutions of sulfuric acid (24,25) and formalin (26) are used to incubate the isolated eggs at incubation temperatures ranging between 22°C and 26°C and duration of the incubation period between 21 and 30 days (26). The solution of sulfuric acid (0.1N) was reported to give the best result, between 75-80% (22) and 83-92% (27) viability, followed by formalin solution (75-80%) (26). ...
... Solutions of sulfuric acid (24,25) and formalin (26) are used to incubate the isolated eggs at incubation temperatures ranging between 22°C and 26°C and duration of the incubation period between 21 and 30 days (26). The solution of sulfuric acid (0.1N) was reported to give the best result, between 75-80% (22) and 83-92% (27) viability, followed by formalin solution (75-80%) (26). The disadvantage of incubation in determining the viability of helminth eggs is the duration of time. ...
According to the World Health Organisation, more than 1.5 billion of the world population is affected by parasitic diseases caused by geohelminths. The number of persons suffering from foodborne and waterborne protozoan diseases is similar. In developed countries, including Bulgaria, systematic sanitary and parasitological studies of soil and water are the basis for monitoring and control in protecting public health. Occurrence and prevalence of human parasitic infections is determined by the peculiarities of life cycle of parasites, peculiarities of hosts as reservoir sources, the abiotic and biotic factors of the environment as a complex providing conditions for the development or sterilisation of different parasite stages, as well as socioeconomic factors that play a leading role in the whole epidemiological process.
Systematic sanitary-parasitological studies require the application of classical and novel reliable, sensitive and practical diagnostic methods that are also easy to perform, economical and efficient enough.
... Moreover, a comparative analysis of the studies listed in Table 2 confirms these assumptions by showing that Tween®80, glycine and 7X® seem to be superior to other detergents as indicated by higher recovery rates when these agents were used (Fig. 3a). Moreover, Tween®20/40/80 or 7X® have been used by numerous investigators as both are soluble in water at any given concentration and 7X® does not form precipitates with highly concentrated salts used for flotation (Bowman et al., 2003). ...
... After dissociation of eggs from larger organic and inorganic particles, separation of eggs from particles is commonly conducted via filtration through sieves (Bowman et al., 2003;Engohang-Ndong et al., 2015;Katakam et al., 2014), in which the choice of pore size is crucial to optimise egg recovery from different sample matrices. More precisely, ascarid eggs have varying dimensions ranging from approximately 45-75 μm for Ascaris spp., 45-90 μm for Ascaridia galli, 45-75 μm for Heterakis gallinarum, 90-100 μm for Parascaris spp., 60-95 μm for Toxocara spp. ...
... Nevertheless, adverse effects of high-density flotation solutions like viscosity or chemical interactions with the eggs' outer surfaces proteins seem to play an eminent role in recovery. For instance, the osmotic pressure might result in distortion of eggs (Steinbaum et al., 2017) or the viscosity of a sucrose solution may interfere with the flotation speed of eggs (Bowman et al., 2003). ...
Ascarid infections constitute a major concern for both human and animal health risk assessment. Although being effectively transmitted by soil, water and contaminated food, reliable detection of ascarid eggs in environmental media often remains challenging. However, contamination of the environment with ascarid ova has gained more attention as a decisive part of proper risk assessment in recent years. Due to various factors, such as sample matrices, dissociation detergents and flotation solutions, defined and standardised protocols for the isolation of eggs from complex environmental matrices are difficult to establish and therefore limited. Thus, this study reviews common techniques used for the recovery of ascarid eggs from environmental media with special emphasis on sampling strategies, purification procedures and microscopic as well as molecular detection of egg contamination. Despite various advancements, mainly in the field of molecular methods leading to more reliable and sensitive detection, it can be concluded that there is still a need for unified guidelines for sampling and recovery of ascarid eggs derived from complex environmental matrices.
... As a result of their environmental hardiness, the WHO recommends parasitic helminth ova as an indicator of sanitary risk and water quality parameters [19,20]. WHO recommends an upper limit of one helminth ova per litre for recycled water to be judged suitable for irrigation and public use [2,21,22]. Based on such recommendations, the modified Bailenger method was suggested as a universal method for the detection of one helminth ova per 10 litres of recycled water for urban, agricultural, industrial, or environmental use [23,24]. However, the method has drawbacks, such as being time consuming (approximately three days), insensitive, and involves ova recovery, morphological identification, and enumeration of helminth ova [25][26][27]. ...
... The A. suum infected pig faecal samples were obtained from an abattoir in Laverton, Australia. The ova of A. suum were recovered using a modified version of the Tulane method [21,52]. Aliquots of pig faecal samples with approximately 5 g DS (dissolved solid) were rinsed with milliQ water, homogenised for 1 min with a blender, and allowed to settle in 1% 7X detergent for 30 min (MP Bio, Australia). ...
... Faecal samples were collected from pigs that were infected with T. suis. The ova were recovered using Tulane method with minor modifications (21,52) and were also used for further experiments. Genomic DNA of A. lumbricoides and H contortus were obtained from the Faculty of Veterinary and Agricultural Sciences, The University of Melbourne. ...
Ascaris lumbricoides is a major soil-transmitted helminth that is highly infective to humans. The ova of A. lumbricoides are able to survive wastewater treatment, thus making it an indicator organism for effective water treatment and sanitation. Hence, Ascaris ova must be removed from wastewater matrices for the safe use of recycled water. Current microscopic techniques for identification and enumeration of Ascaris ova are laborious and cumbersome. Polymerase chain reaction (PCR)-based techniques are sensitive and specific, however, major constraints lie in having to transport samples to a centralised laboratory, the requirement for sophisticated instrumentation and skilled personnel. To address this issue, a rapid, highly specific, sensitive, and affordable method for the detection of helminth ova was developed utilising recombinase polymerase amplification (RPA) coupled with lateral flow (LF) strips. In this study, Ascaris suum ova were used to demonstrate the potential use of the RPA-LF assay. The method was faster (< 30 min) with optimal temperature at 37 °C and greater sensitivity than PCR-based approaches with detection as low as 2 femtograms of DNA. Furthermore, ova from two different helminth genera were able to be detected as a multiplex assay using a single lateral flow strip, which could significantly reduce the time and the cost of helminth identification. The RPA-LF system represents an accurate, rapid, and cost-effective technology that could replace the existing detection methods, which are technically challenged and not ideal for on-site detection in wastewater treatment plants.
... 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. ...
... Samples of raw wastewater and sludge were collected from two different wastewater treatment plants (Lang Lang and Blind Bight) run by South East Water, Victoria, Australia. Because no indigenous Ascaris ova were observed in the aforementioned samples while analysed using the US EPA recommended Tulane method [10], the samples were spiked with Ascaris suum ova for the initial experiment investigating the impact of modifications in the Tulane method (Table 1) on the recovery efficiency of A. suum. The ova of A. suum were recovered from infected pig faecal samples and were used as a surrogate for A. lumbricoides due to their lower infectivity to humans, despite being identical morphologically and exhibiting 98.1% genetic similarity to A. lumbricoides [12,13]. ...
... The standard Tulane method [10] consisted of a settling volume of 900 mL, settling time of approximately 240 min with a single centrifugal flotation at 800 g for 10 min. Table 1 indicates the parameters that were modified from the original Tulane method in order to optimise parameters leading to an increased yield of ova recovery whilst minimising processing time. ...
... These methods do not provide viability status of helminth ova. In view of this, culture and vital stain methods have been commonly used to quantify the viable helminth ova from wastewater and sludge (Bowman et al., 2003;de Victorica and Galván, 2003;US EPA, 2003;Trang et al., 2006;Do et al., 2007;Wen et al., 2009;Sharafi et al., 2012). However, there are some significant limitations of the culture and vital stain methods. ...
... Several methods such as centrifugation (Whitmore and Carrington, 1993;Higgins et al., 2003), hollow fiber ultrafiltration (HFUF) (Simmons et al., 2001;Ferguson et al., 2004;Hill et al., 2005;Hill et al., 2007), filtration (Nieminski et al., 1995;Maya et al., 2006;Alli et al., 2011), and flotation (Bowman et al., 2003;de Victorica and Galván, 2003;Bastos et al., 2013) have also been used to recover various microorganisms including ova from water and soil samples. Some of these methods are rapid and can potentially be used to concentrate helminths ova from wastewater matrices. ...
... The culture-based method involves artificially hatching the ova in a laboratory. Helminth ova are incubated at 28ºC -30ºC for up to 28 days depending on the helminths, to allow the viable ova to hatch and are observed microscopically (Bowman et al., 2003). Health regulators including the US EPA and WHO recommend this method because it has the ability to estimate viability of helminth ova recovered from treated wastewater and sludge. ...
... The choice of technique used is largely influenced by the different types of samples (Maya et al., 2006). One reason for this may be the lack of published quality assurance/quality control (QA/QC) data on the various methods (Bowman et al., 2003). In addition to the more traditional methods based on sedimentation and/or flotation, which mainly involves the separation and concentration of eggs and the microscopical identification and quantification of these eggs, several new techniques have been developed. ...
... These methods recommend the use of one liter samples ( Molleda et al., 2008). Furthermore, the volume of water analyzed is directly related to the level of detection achieved (Bowman et al., 2003). For instance, the WHO guidelines for wastewater reuse in agriculture recommends <1 viable helminth egg/Liter for unrestricted irrigation (WHO, 2006). ...
... Table 2 shows concentration of eggs in sludge from different locations, with a higher concentration in sludge samples from developing countries irrespective of the sample size. The amount of sludge sampled is often related to the dry weights, which are in the interval from 2 g (Scaglia et al., 2014;Pecson et al., 2007;Nelson and Darby, 2001) to 5 g (Bowman et al., 2003). This contrasts with the USEPA method that recommends 300 g as a dry weight sample size (Cappizzi-Banas et al., 2004;Maya et al., 2012;Gantzer et al., 2001). ...
It is estimated that over a billion people are infected with soil-transmitted helminths (STHs) globally with majority occurring in tropical and subtropical regions of the world. The roundworm (Ascaris lumbricoides), whipworm (Trichuris trichiura), and hookworms (Anclostoma duodenale and Necator americanus) are the main species infecting people. These infections are mostly gained through exposure to faecally contaminated water, soil or contaminated food and with an increase in the risk of infections due to wastewater and sludge reuse in agriculture. Different methods have been developed for the detection and quantification of STHs eggs in environmental samples. However, there is a lack of a universally accepted technique which creates a challenge for comparative assessments of helminths egg concentrations both in different samples matrices as well as between locations. This review presents a comparison of reported methodologies for the detection of STHs eggs, an assessment of the relative performance of available detection methods and a discussion of new emerging techniques that could be applied for detection and quantification. It is based on a literature search using PubMed and Science Direct considering all geographical locations. Original research articles were selected from the results and the methodology sections and assessed in relation to the methods used. Methods reported in these articles were grouped into conventional, molecular and emerging techniques, the main steps in each method were then compared and discussed. The inclusion of a dissociation step aimed at detaching helminth eggs from particulate matter was found to improve the recovery of eggs. Additionally the selection and application of flotation solutions that take into account the relative densities of the eggs of different species of STHs also results in higher egg recovery. Generally the use of conventional methods was shown to be laborious and time consuming and prone to human error. The alternate use of nucleic acid-based techniques has improved the sensitivity of detection and made species specific identification possible. However, these nucleic acid based methods are expensive and less suitable in regions with limited resources and skill. The loop mediated isothermal amplification method shows promise for application in these settings due to its simplicity and use of basic equipment. In addition, the development of imaging soft-ware for the detection and quantification of STHs shows promise to further reduce human error associated with the analysis of environmental samples. It may be concluded that there is a need to comparatively assess the performance of different methods to determine their applicability in different settings as well as for use with different sample matrices (wastewater, sludge, compost, soil, vegetables etc.).
... methods to extract and concentrate Ascaris spp. ova from biosolids via flotation and sedimentation have proven to be adequate [16][17][18][19]. however, there is not an universally accepted assay for determining viability of ova [16]. ...
... ova from biosolids via flotation and sedimentation have proven to be adequate [16][17][18][19]. however, there is not an universally accepted assay for determining viability of ova [16]. Staining techniques are rapid, as viable ova contain multiple layers that are impermeable, whereas ova that are permeable are assumed to be non-viable [20][21][22]. ...
... however, this method may be subjective as it incorporates total nucleic acid, without any discrepancy for infectious and non-infectious ova. current microscopy methods dictate viable ova as those containing motile distinguishable larvae, but all others are non-viable [16,[24][25][26][27] [17]. yet, these methods are subject to major constraint, as they do not monitor embryo development, and/or consider all ova displaying early-stages of embryonation, prior to motile larvae, to be non-viable. ...
This study suggests a new method for determining the viability of Asca-ris spp. ova, based on in-vitro early-to-late stage development of ova. This method includes stages prior to larval development, providing an estimation of potential viability. After application of biosolids onto soil and exposure to 7°C, 22°C, or 37°C for 45 days, ova were microscopically distinguished as viable or non-viable according to progression through development categories. Results were compared to viability estimates from current methods that distinguish viable ova as motile larva. Results suggest conventional techniques underestimate viability, whereas the new method provides a more conservative approach.
... Sludge samples that do not represent the actual diversity of the environment entail in the disqualification of any further result obtained by the application of a certain method (USEPA, 2003). Furthermore, the amount of sludge that is used in the quantification assays relates directly to the level of detection achieved (Bowman et al., 2003). ...
... However, as a primary strategy to remove the eggs from the sludge matrix washing of the samples with solutions of surfactants is recommended. According to Bowman et al. (2003), non-ionic detergents, such as Triton ® , Tween ® 80 and 7X ® are commonly used as surfactants. On one hand, low concentrations of these substances may effectively desorb the eggs from the sludge (Reimers et al., 1989;Gaspard et al., 1996;Bowman et al., 2003). ...
... According to Bowman et al. (2003), non-ionic detergents, such as Triton ® , Tween ® 80 and 7X ® are commonly used as surfactants. On one hand, low concentrations of these substances may effectively desorb the eggs from the sludge (Reimers et al., 1989;Gaspard et al., 1996;Bowman et al., 2003). On the other hand, due to probable damage to the integrity of the membrane of the egg, rinsing the sludge with high concentrations of a detergent may cause a toxic effect on helminth eggs. ...
For the application of sewage sludge as fertilizer, it is of fundamental importance the absence of pathogenic organisms, such as viable helminth eggs. Thus, the quantification of these organisms has to be carried out by means of the application of reliable and accurate methodologies. Nevertheless, until the present date, there is no consensus with regard to the adoption of a universal methodology for the detection and quantification of viable helminth eggs. It is therefore necessary to instigate a debate on the different protocols currently in use, as well as to assemble relevant information in order to assist in the development of a more comprehensive and accurate method to quantify viable helminth eggs in samples of sewage sludge and its derivatives.
... For the PMA-qPCR method development, A. caninum ova were used as a representative for human hookworm. A. caninum ova were isolated from dog fecal samples collected from the School of Veterinary Science, University of Queensland, Australia, using a flotation method (Bowman et al. 2003). The numbers of A. caninum ova were estimated by microscopic observation. ...
... The tube was further centrifuged at 5200g for 10 min to obtain a pellet. Ova were separated from the pellet using the flotation method (Bowman et al. 2003). Separated ova were then treated with 100 μM PMA. ...
... The samples were mixed with potassium carbonate (K 2 CO 3 ) and transported on ice to Australia for laboratory analysis. Upon arrival, ova were recovered from human fecal and soil samples using the floatation method (Bowman et al. 2003). ...
... For the PMA-qPCR method development, A. caninum ova were used as a representative for human hookworm. A. caninum ova were isolated from dog fecal samples collected from the School of Veterinary Science, University of Queensland, Australia, using a flotation method (Bowman et al. 2003). The numbers of A. caninum ova were estimated by microscopic observation. ...
... The tube was further centrifuged at 5200g for 10 min to obtain a pellet. Ova were separated from the pellet using the flotation method (Bowman et al. 2003). Separated ova were then treated with 100 μM PMA. ...
... The samples were mixed with potassium carbonate (K 2 CO 3 ) and transported on ice to Australia for laboratory analysis. Upon arrival, ova were recovered from human fecal and soil samples using the floatation method (Bowman et al. 2003). ...
In this study, we have evaluated the efficacy of Propidium Monoazide (PMA) to differentiate between viable and non-viable Ancylostoma caninum ova. The newly developed method was validated using raw wastewater seeded with known numbers of A. caninum ova. Results of this study confirmed that PMA-qPCR has resulted in average of 88% reduction (P < 0.05) in gene copy numbers for 50% viable + 50% non-viable when compared with 100% viable ova. 100% reduction in gene copies was observed for 100% non-viable ova when compared with 100% viable ova. Similar reductions (79-80%) in gene copies were observed for A. caninum ova seeded raw wastewater samples (n = 18) collected from wastewater treatment plant (WWTP) A and B. The newly developed PMA-qPCR method was applied to determine the viable ova of different helminths (A. caninum, A. duodenale, Necator americanus and Ascaris lumbricoides) in raw wastewater, human fecal and environmental soil samples. None of the unseeded wastewater samples were positive for above mentioned helminths. Ova of Necator americanus and Ascaris lumbricoides were found in unseeded human fecal and environmental soil samples. For the unseeded human fecal samples (1 gm), average gene copy concentration obtained from qPCR and PMA-qPCR was found to be similar (6.8 × 105 ± 6.4 × 105 and 6.3 × 105 ± 4.7 × 105) indicating the presence of viable N. americanus ova. Among the 24 unseeded soil samples tested, only one was positive for A. lumbricoides. The mean gene copy concentration in the positively identified soil sample was 1.0 × 105 ± 1.5 × 104 (determined by qPCR) compared to 4.9 × 104 ± 3.7 × 103 (determined by PMA-qPCR). The newly developed PMA-qPCR methods were able to detect viable helminth ova from wastewater and environmental samples and could be adopted for health risk assessment.
... Some studies have aimed to detect Toxocara spp. and other soil-transmitted helminths in soil samples [12,13]; however, the two such studies conducted in the United States sampled sewage sludge rather than soil [12,14]. Furthermore, these studies utilized conventional microscopy-based detection of helminth eggs, which are subjective and inaccurate. ...
... Nonetheless, as soil-transmitted helminths, they can also be ingested from the soil by children. Furthermore, the burdens of these parasites in soil have been associated with their prevalences in their sampling areas [14]. ...
Background
Gastrointestinal parasites are generally associated with lower-income countries in tropical and subtropical areas, but they are also prevalent in low-income and extreme low-income communities in the Southern United States. To date, studies characterizing the epidemiology of gastrointestinal parasites in the United States are limited, resulting in little comprehensive understanding of the challenge. This study investigates the environmental contamination of gastrointestinal parasites in the Southern United States by determining the contamination rate and burden of each parasite in five low-income communities.
Methods
A total of 499 soil samples of approximately 50g were collected from public parks and private residences in Alabama, Louisiana, Mississippi, South Carolina, and Texas. A novel technique utilizing parasite floatation, filtration, and bead-beating was applied to concentrate and extract parasite DNA from samples and detected via multi-parallel qPCR.
Findings
qPCR detected Blastocystis spp (19.0%), Toxocara cati (6.01%), Toxocara canis (3.61%), Strongyloides stercoralis (2.00%), Trichuris trichiura (1.80%), Ancylostoma duodenale (1.42%), Giardia intestinalis (1.40%), Cryptosporidium spp (1.00%), Entamoeba histolytica (0.201%), and Necator americanus (0.200%). Overall parasite contamination rates varied significantly between communities: Western Mississippi (46.88%); Southwestern Alabama (39.62%); Northeastern Louisiana (28.24%); Southwestern South Carolina (27.03%); and South Texas (6.93%) (p < 0.0001). Toxocara cati DNA burdens were greater in communities with higher poverty rates, including Northeastern Louisiana (50.57%) and Western Mississippi (49.60%) compared to Southwestern Alabama (30.05%) (p = 0.0011).
Interpretation
This study demonstrates the environmental contamination of parasites and their relationship with high poverty rates in communities in the Southern United States.
Funding
This work was supported by the Maternal and Infant Environmental Health Riskscape (MIEHR) Center of Excellence on Environmental Health Disparities Research, NIMHD grant #P50 MD015496
Research in context
Evidence before this study
Several research articles on parasites in the Southern USA were used to determine the extent of parasitosis in the selected regions (Mckenna et al, Am J Trop Med Hyg 2018; Singer et al, Am J Trop Med Hyg 2020; Bradbury et al, Emerg Infect Dis 2021). Criteria used included terms hookworm, soil-transmitted helminths, protozoa, and parasites in the United States. The most recent parasite prevalence studies indicate areas with 62.9% Blastocystis , 34.5% hookworm ( Necator americanus ), 16.5% Strongyloides stercoralis , 5.2% Toxocara , 2.9% Cryptosporidium , 2.3% Giardia intestinalis , and 1.8% Entamoeba histolytica from human serum or stool samples. These studies focus on human parasite infections, but there are no current published studies on environmental parasite surveys in the Southern US. The parasites in this study all have part of their life-cycle in soil and can directly infect humans living in these areas.
Added value of this study
Our work expands the current understanding of prevalence of parasites in the Southern US soil from built environments. We further correlate the type of parasites and their intensity of soil contamination with higher poverty rates.
Implications of all the available evidence
These parasitic infections represent a major source of contamination in the built environments and their association with poverty. This study will help focus public policy on the potential risks that environmental parasites have on human health. It further describes the high contamination rates in the USA, a high-income country.
... The floating eggs are subsequently harvested from the supernatant using sieves of appropriate mesh size and counted via visual identification under a microscope. To test viability, the collected Ascaris eggs can be incubated at 25 • C for up to 28 days, which are optimal conditions for larval development which is ascertained by microscopic examination [11,65]. ...
... The application of the Tulane method has led to egg recovery rates of 60-76% from sludge samples [65,66] and this method is commonly used to recover and enumerate HE in WWTPs in Australia. A study indicated the HE rate of recovery for soil analysis was 75.5% with a precision of 32.5% [67]. ...
Understanding and managing the risk posed by helminth eggs (HE) is a key concern for wastewater engineers and public health regulators. The treatment processes that produce recycled water from sewage at wastewater treatment plants (WWTPs) rely on achieving a defined log10 reduction value (LRV) in HE concentration during the production of recycled water from sewage to achieve the guideline concentration of ≤1.0 HE/L. The total concentration of HE in sewage reaches thousands of HE/L in developing countries and therefore, an LRV of 4.0 is generally accepted to achieve a safe concentration in recycled water, as this will meet the guideline value. However, in many developed countries with good sanitation and public health standards, the HE concentration in sewage is generally <10 HE/L. Therefore, validation of the sewage treatment process relied on to achieve an LRV of 4.0 can be difficult. Because of these limitations, design equations to predict LRVs from hydraulic retention times (HRT), which are geographically non-specific, are commonly relied on to ensure the production of safe quality recycled water with respect to HE. However, these design equations could be further refined by defining the design and management of the treatment process in greater detail and thus be used more effectively for determining the LRV required. This paper discusses the limitations and possible improvements that could be applied to LRV design equations for predicting HE removal at WWTPs and identifies the data requirements to support these improvements. Several options for LRV design equations are proposed that could be validated experimentally or via the ongoing operation of WWTPs. These improvements have the potential to assist the rationalization of the HE removal requirements for specific treatment options, exposure scenarios and use of recycled water in agriculture.
... Regarding the accuracy obtained for the assays, samples from the drying bed that contained sludge from septic tanks, presented an average recovery of A. suum eggs of 74% (±6.1%). This value is in accordance with that expected for the recovery of the eggs, which is between 75% and 80% for anaerobic sludge as verified by Bowman et al. (2003). However, samples collected from the drying bed that contained UASB sludge presented 54% (±4.2) of recovery of the inoculated eggs. ...
... The difference in the recovery of A. suum eggs for sludge samples that have similar characteristics, that is, both anaerobic and disposed of in drying beds, was unforeseen. In fact, when the accuracy of the USEPA protocol was tested by Bowman and colleagues [21], only four matrices of biosolids were evaluated, namely: (1) synox-treated biosolids (acid treatment process); (2) anaerobically treated biosolids stored in lagoons; (3) soil-biosolid blends; and (4) biosolids from Chemfix process (alkaline treatment). However, the USEPA (2003) generalizes the application of this methodology for samples of sewage sludge and compost (Appendix I EPA/625/R-92/013). ...
Two bench scale anaerobic sequencing batch reactors (ASBR) were operated to evaluate the reduction of organic matter and ammonium nitrogen concentrations in landfill leachate. The reactors were inoculated with sludge collected from the bottom of a facultative pond, located at Curitiba's Sanitary Landfill, Parana, Brazil. The biomass of one of the reactors was bioaugmented by the addition of anaerobic microorganisms cultured in a medium containing 20 mL of raw landfill leachate and agar as a gelling agent. The microorganisms, isolated from the leachate were processed and visualized by scanning electron microscopy, using a novel preparation technique, which includes the growth of the microorganisms on the surface of a membrane of cellulose nitrate. Microscopic and molecular analysis of the cultures indicated that hydrogenotrophic microorganisms were the majority of the methanogen population, cultivated from samples of landfill leachate. The reactor with no augmentation presented removal efficiencies of COD, DOC and N-NH3 concentrations of 40%, 70% and 20%, respectively, whereas the reactor with augmentation presented efficiencies of 60%, 75% and 21%, respectively. Furthermore, it has been found out that the structure of the granules produced in the reactor with bioaugmented biomass was more uniform, even after two months from the addition of the microbial inoculum. The production of higher extracellular polymeric substance (EPS) may explain the structural stability of the granules in the system which received bioaugmentation, however, further research should be carried out to confirm these results.
... Regarding the accuracy obtained for the assays, samples from the drying bed that contained sludge from septic tanks, presented an average recovery of A. suum eggs of 74% (±6.1%). This value is in accordance with that expected for the recovery of the eggs, which is between 75% and 80% for anaerobic sludge as verified by Bowman et al. (2003). However, samples collected from the drying bed that contained UASB sludge presented 54% (±4.2) of recovery of the inoculated eggs. ...
... The difference in the recovery of A. suum eggs for sludge samples that have similar characteristics, that is, both anaerobic and disposed of in drying beds, was unforeseen. In fact, when the accuracy of the USEPA protocol was tested by Bowman and colleagues [21], only four matrices of biosolids were evaluated, namely: (1) synox-treated biosolids (acid treatment process); (2) anaerobically treated biosolids stored in lagoons; (3) soil-biosolid blends; and (4) biosolids from Chemfix process (alkaline treatment). However, the USEPA (2003) generalizes the application of this methodology for samples of sewage sludge and compost (Appendix I EPA/625/R-92/013). ...
The application of sewage sludge on arable and grassland soils is dependent on the assurance that the concentrations of pathogens present in the biosolids do not pose health risks to either individuals or animals. Therefore, the use of appropriate protocols for the detection and quantification of pathogens in sludge samples is essential. An ideal method for the quantification should provide high sensitivity, accuracy and precision, but shall also follow a program that ensures the quality of the data produced. Methodologies that ensure accurate and reliable responses in a short period of time are also desirable. Therefore, this work aimed at the assessment of the accuracy of the method specified by the U.S. Environmental Protection Agency (USEPA) and used for the quantification of viable Ascaris eggs eventually present in biosolids. This study was carried out using samples of anaerobic sewage sludge collected from two drying beds, one receiving sludge from an upflow anaerobic sludge blanket reactor, and the other receiving sludge from a septic tank.
Keywords: anaerobic sludge; Ascaris suum; helminths; parasitology of sludge, quantification methods
... However, a major drawback of this study was the lack of reliability of the microscopic evaluations for accurate identification of the late-morula and blastula stages due to more time and higher magnification required. Due to the accurate identification of viable early embryonic stages, the present study was successful in lowering the time required for viability testing in comparison to the standard Tulane method [73]. ...
Throughout history, parasites and parasitic diseases have been humankind’s constant companions, as evidenced by the findings of tapeworm eggs in ancient, mummified remains. Helminths are responsible for causing severe, long-term, and debilitating infectious diseases worldwide, especially affecting economically challenged nations due to prevailing deficits in access to sanitation, proper hygiene practices, and healthcare infrastructure. Socio-ecological drivers, such as poverty, migration, and climate change, continue to contribute to parasites and their disease vectors being spread beyond known endemic zones. The study of parasitic diseases has had a fair amount of success leading to the development of new chemotherapeutic agents and the implementation of parasite eradication programs. However, further progress in this direction has been hampered by the challenges of culturing some of these parasites in in vitro systems for efficient availability, basic life cycle, infection studies, and effectiveness of novel treatment strategies. The complexity of the existing models varies widely, depending on the parasite and its life cycle, ranging from basic culture methods to advanced 3D systems. This review aims to highlight the research conducted so far in culturing and maintaining parasites in an in vitro setting, thereby contributing to a better understanding of pathogenicity and generating new insights into their lifecycles in the hopes of leading to effective treatments and prevention strategies. This work is the first comprehensive outline of existing in vitro models for highly transmissible helminth diseases causing severe morbidity and mortality in humans globally.
... The most commonly used detergent in helminth methodsused in the US EPA Method; also used in tissue culture and is low foaming. Bowman et al., 2003;US EPA, 2003;Capizzi-Banas et al., 2004;Maya et al., 2012;Pecson et al., 2007; PRG, 2017 ...
This technical report highlights the experiments and steps involved in the comparison of existing helminth test methods for water and sanitation samples, and the subsequent compilation of the most successful parameters to form an optimised method.
... The same analytical methods as used for the water samples were applied to the soil and manure samples, but modified to suit solid samples according to the procedure described by Verbyla et al. (2016) for E. coli and coliphages, and complemented with the Tulane method for helminth eggs (Bowman et al., 2003). For analysis of E. coli and coliphages, 25 g of soil sample were added to 225 mL sterile water and shaken vigorously for 1 m, and the mixture was then processed as described for water samples (see Section 2.4.2). ...
On-farm measures can be used in multi-barrier schemes to manage microbial risks from consumption of wastewater-irrigated vegetables, especially where informality of the practice determines minimal external support for farmers. Evidence indicates that cessation of irrigation greatly reduces microbial contamination on leafy vegetables, but at the expense of produce quality. Replacing wastewater with higher-quality irrigation water during the last days of cultivation is an alternative to cessation of irrigation that does not compromise produce quality. This study evaluated the effect of wastewater substitution under on-farm conditions on different indicators of microbial contamination of lettuce. Lettuce was cultivated in experimental plots and irrigated with three water sources: spring water, water from a wastewater-polluted river and effluent from a primary wastewater treatment plant, but with the river water replaced by spring water in half the plots about two weeks before harvest. The experiment was repeated four times in different seasons. Irrigation water samples collected during cultivation and lettuce samples collected at harvest were analysed for helminth eggs, Escherichia coli and coliphages. Variables characterizing the irrigation practices and environmental conditions were recorded. There were no significant differences in helminth egg or E. coli concentrations on lettuce (medians ranged from −0.7 to −0.1 log10 eggs g⁻¹ and 0.6–1.4 log10 cfu g⁻¹, respectively) between any of the treatments involving wastewater irrigation; no statistical analysis was possible for coliphages because concentrations on lettuce were mostly at or below the detection limit (94% of samples). Variables associated with temperature and soil explained helminth egg and E. coli concentrations on lettuce, while number of days of irrigation with spring water (representing wastewater substitution) was significant only for E. coli. It was concluded that the experimental conditions were suboptimal for successful implementation of wastewater substitution for on-farm microbial risk management, but key variables for successful implementation were identified.
... To quantify helminth eggs in each stage of solar drying process for limed and non-limed sludge, the flotation method adapted for biowaste analysis (Bowman et al. 2003;US EPA Protocol 1999) and modified by Koné et al. (2007) and Schwartzbrod (2003) was applied. This analytical technique was carried out on 5 g of fresh SS (an average of 30% of DM). ...
The present study is aimed at assessing the effectiveness of solar drying process in terms of helminth egg reduction in sewage sludge (SS) generated from an activated sludge wastewater treatment plant (WWTP) in Marrakesh city (Morocco). It is also engaged to highlight a synergic effect of liming (1% CaO) and solar drying on helminth egg reduction. The solar drying process was conducted for 45 days, in summer under a semi-arid climate in a pilot scale polycarbonate-based tunnel (2 m³). Before undergoing solar drying process, data showed an important load of helminth eggs including Ascaris sp., Schistosoma spp., Capillaria spp., Trichuris spp., Ankylostome spp., Toxocara spp., and Taenia spp. in limed sludge (LS) and non-limed sludge (NLS) (15.2 and 17.9 eggs/g, respectively). Ascaris eggs were the most abundant (11.2 and 13.5 eggs/g in LS and NLS, respectively). By the end of the solar drying process, a considerable removal of the total helminth eggs was recorded in LS and NLS (92.8% and 91.6%, respectively). A complete removal of Schistosoma spp., Capillaria spp., Trichuris spp., Toxocara spp. and Taenia spp. was noted in LS and NLS. In the case of Ankylostome spp., data showed a total removal in LS and 81% in NLS; however, the final load is in agreement with the standards (0.4 egg/g). As for Ascaris spp., neither liming nor solar drying process allowed a complete removal (91% and 90% in NLS and LS, respectively) and the final load (1.1 egg/g) does not fulfill the WHO requirements for an agricultural use. Principal component analysis (PCA) demonstrated a negative correlation between dry matter (DM) content (hence temperature) and helminth egg concentration. No significant synergic effect of liming and solar drying process was showed by statistical analysis. This is substantiating that temperature is the key parameter involved in helminth egg removal while undergoing solar drying of SS.
... One plastic chamber from each media bottle was removed during sampling on Day 6 and 15. Ascaris eggs were recovered from the plastic chambers using a method adapted from several methods published previously (Nelson and Darby, 2002;Bowman et al., 2003;Trönnberg et al., 2010;Amoah et al., 2018). First, the mesh from one side was peeled off, and a metal spatula was used to divide most of the contents between two 50-mL centrifuge tubes. ...
Innovative, low-cost methods for inactivating pathogens in human fecal material (HFM) are needed, particularly in expanding urban areas where conventional sewer systems and centralized wastewater treatment plants are not feasible. To address this challenge, we have developed a bioprocess that utilizes open cultures of anaerobic bacteria (i.e., microbiomes) to produce carboxylic acids using HFM as substrate. When the pH is sufficiently low, the carboxylic acids exist in the undissociated form and inactivate pathogens. Here, we used real solid waste (SW) collected from container-based, urine-diverting dry toilets (UDDT) in Nairobi, Kenya to conduct lab-scale and field-scale trials. Through these trials, we investigated operating conditions required to use carboxylic acid fermentation in sanitation waste treatment processes. We tested three different inoculum treatments and determined that the microbiome in UDDT-SW is well-suited to produce carboxylic acids without the need for an external inoculum. We also tested co-fermentation of UDDT-SW with carbohydrate-rich food waste as a means of reducing the pH. We found that when food waste was incorporated in a way that maintained the pH between 4.8 and 5.2, then the food waste was quickly converted to carboxylic acids, and the low pH created high concentrations of undissociated carboxylic acids. The resulting concentrations of undissociated carboxylic acids resulted in Ascaris inactivation within 15 days. However, we found that a temperature ≥30°C is required for carboxylic acid production to occur.
... The methods used for the examination of sewage sludge are mainly those developed for the examination of soil (Quinn et al., 1980;Polish Standardisation Committee, 2001;Ayres and Mara, 1996;US EPA, 1999;Nelson and Darby, 2001;Bowman et al., 2003). ...
The use of sewage sludge in agriculture brings the risk of microbiological and parasitological contamination of soil, ground and surface water, as well as cultivated plants. Therefore prior the application to the soil, sewage sludge must be examined, among others, for the presence of live eggs of intestinal parasites. However, the efficiency of commonly used for this purpose parasitological methods is not satisfactory. This is probably due to the presence of flocculants in the sediments used in the dehydration process. The objective of the study was analysis of the effect of flocculant (cationic acrylamide) on the possibilities of isolation of parasite eggs from dehydrated sewage sludge. For this purpose 10 samples of sewage sludge were prepared: 5 containing flocculant and 5 without flocculant. Samples were tested by flotation method according to Quinn. From sewage sludge free of flocculant, 67.8 eggs were isolated, on average, whereas from sludge containing flocculant – only 2.8 eggs. The experiments confirmed that the isolation of eggs from sewage sludge containing flocculant (cationic acrylamide) is much more difficult than from sludge free from this substance and therefore the simple parasitological methods should not be used to examine the dehydrated sewage sludge.
... For this reason, the analysis of the helminth eggs was carried out on 5 g of fresh matter. The concentration of the parasitic elements was achieved according to the applied flotation method for the analyses of biowastes (Bowman et al. 2003; US EPA Protocol 1999) modified by Schwartzbrod (2003); Koné et al. (2007). After particles scattering by ammonium bicarbonate (11.9%) for few minutes, and because of the weaker density of Ascaris and trichuris eggs, 1.10 and 1.15 respectively (David and Lindquist 1982), and after centrifugation to 1389 g/3 min, the pellet was re-suspended in 40 mL of ZnSO 4 (56.81%), after 10 min, the mixture was centrifuged at 617 g/ 3 min, and the supernatant was recovered in several centrifuge tubes sand washed by distilled water, then centrifuged at 964 g/3 min. ...
The prevalence and the identification of the helminth eggs load of raw sewage sludge was assessed of three different wastewater treatment systems. The results showed a variety of parasite species with following average concentrations; five taxa belonging to three classes nematodes, cestodes and trematodes were inventoried. The class of nematodes is the most diverse with 5 taxa. It is represented by the eggs of Ascaris sp., Capillaria sp., Trichuris sp., Toxocara sp., and Ankylostome sp., then comes the cestodes class, this is represented by the eggs of Tænia sp. The trematode class is represented by Schistosoma sp. The lagooning station of Chichaoua shows the highest load 7 species with Ascaris 21 eggs/g; Capillaria sp., 11 eggs/g; Trichuris sp., 6 eggs/g; Toxocara sp., 2 eggs/g and Ankylostome sp., 1 egg/g; Taenia sp., 2eggs/g; and Schistosoma sp., 1 egg/g. Infiltration-percolation sludge show the presence of 4 species of helminths eggs in sludge from anaerobic settling with different rates: 15 eggs/g for Ascaris sp., 15 eggs/g for Trichuris sp., 13 eggs/g for Capillaria sp., and 8 eggs/g for Taenia sp. However, in sand filter pool, the sludge helminth eggs load was decreased by 47% of Ascaris sp., 85% of Capillaria sp., and 75% of Taenia sp., Nevertheless, an increase of Trichuris eggs load was noted in the second sludge by 17%. Five helminth eggs was detected in primary sludge coming from decantation pools in activated sludge plant in Marrakech, that is Ascaris sp., with a load of 16 eggs/g; Capillaria sp., with 3 eggs/g, Trichuris eggs with 2 eggs/g; Taenia sp., with 4 eggs/g; and Schistosoma sp., with 2 eggs/g. The abatement load of Ascaris sp. with 81% and Schistosoma and Taenia sp., with 100% was noted in biological sludge. Nevertheless, an increase load of Capillaria and Trichuris eggs 81% and 75% respectively was observed in this sludge coming from biological pools. The distribution of parasitic helminth eggs is linked to the differences in demographic and socio-economic status, seasonal variation, physico-chemical characteristic of helminth eggs, and the purification wastewater system performance.
... The second step was the concentration of eggs based on flotation method (Bailenger method) with the presence of zinc sulfate (56.81%, specific gravity, 1.29) (Bowman et al. 2003;Schwartzbrod 2003;Koné et al. 2007;El Fels et al. 2014b). After centrifugation, the liquid supernatant was recovered. ...
In this work, we assessed the drying and composting effectiveness of helminth eggs removal from sewage sludge of a lagoon wastewater treatment plant located in Chichaoua city. The composting was run after mixing sludge with green waste in different proportions: M1 (½ sludge + ½ green waste), M2 ( 2/3 sludge + 1/3 green waste), and M3 ( 1/3 sludge + 2/3 green waste) for 105 days. The analysis of the dewatered sewage sludge showed a load of 8–24 helminth eggs/g of fresh matter identified as Ascaris spp. eggs (5–19 eggs/g) followed by Toxocara spp. (0.2 to 2.4 eggs/g); Hookworm spp. and Capillaria spp. (0.4–1 egg/g); Trichuris spp., Taenia spp., and Shistosoma spp. (< 1 egg/g) in the untreated sludge. After 105 days of treatment by composting, we noted a total reduction of helminth eggs in the order of 97.5, 97.83, and 98.37% for mixtures M1, M2, and M3, respectively. The Ascaris spp. eggs were reduced by 98% for M1 and M3 treatments and by 97% for M2 Treatment. Toxocara spp., Hookworm spp., Trichuris spp., Capillaria spp., and Shistosoma spp. eggs were totally eliminated (100% decrease) and the Taenia spp. was absent from the first stage of composting. These results confirm the effectiveness of both dehydrating and composting processes on the removal of helminth eggs.
... The number of eggs of Ascaris spp. and Taenia spp. in the samples were determined in the laboratory using a modification of the method ofBowman et al. (2003;Shahsavari et al., 2017). The limit of detection was 1 HE/L. ...
The LRVs required to decrease HE concentrations in raw sewage to an acceptable level to manage the risk to human and livestock health were determined. An LRV of 3.0 was required to meet the HBT of 1 μDALY pppy in SE Australia where human helminth infections are not endemic. In comparison, a similar exposure volume and LRV in endemic regions would result in a HBT of 100 μDALY pppy. The risks posed by cattle- and pig-related helminths were also managed acceptably with the treatment of sewage providing an LRV of 3.0. New design equations were derived to determine LRVs based on hydraulic residence times (HRTs) in an activated sludge plant (ASP) and lagoons. The new equation for lagoons indicated that an LRV of 3.0 could be achieved with a HRT of 18 days or less.
... The greater mean percentage recovery of helminth eggs following the modified Bowman method compared to the WHO method was statistically significant (p-value < 0.0001, paired T-test), which demonstrates that the modified Bowman method gives more accurate results in evaluating the concentration of helminth eggs from raw sewage. The method described by Bowman et al. [1] was optimized for the concentration and enumeration of helminth eggs in raw sewage with a significantly improved egg recovery rate compared to the WHO method [2]. The modified Bowman method is a simple, reproducible and low cost method for routine use by commercial laboratories and researchers. ...
Soil-transmitted helminths (STHs) pose a significant public health problem, infecting approximately 2 billion people globally. Despite relatively low prevalence in developed countries, the removal of STHs from wastewater remains crucial to allow the safe use of biosolids or recycled water for agriculture. Wastewater helminth egg count data can contribute to an assessment of the need for, or success of, a parasite management program. Although the World Health Organisation (WHO) has recommended a standard method for counting helminth eggs in raw sewage based on the method of Bailenger (Ayres et al., 1996), the method generally results in low percentage egg recoveries. Given the importance of determining the presence of STHs, it is essential to develop novel techniques that optimise the recovery rate of eggs from raw sewage. In the present study:
• The method described by Bowman et al. (2003) was optimized for the concentration and enumeration of helminth eggs in raw sewage from municipal sewage treatment plants.
• The method is simple and reproducible and recovers a greater percentage of helminth eggs compared to the WHO method.
... Tween 80 is a nonionic, viscous surfactant and 7X is an anionic, non-viscous surfactant. 1% 7X was shown to more efficiently recover Ascaris from biosolids and hands in hand rinse samples than 0.1% Tween 80 [33,34]. We also tested the use of a mixing step where we mixed the sample in a 1000 mL beaker with surfactant up to 600 mL for 10 minutes using a stir plate and magnetic stir bar. ...
Globally, about 1.5 billion people are infected with at least one species of soil-transmitted helminth (STH). Soil is a critical environmental reservoir of STH, yet there is no standard method for detecting STH eggs in soil. We developed a field method for enumerating STH eggs in soil and tested the method in Bangladesh and Kenya. The US Environmental Protection Agency (EPA) method for enumerating Ascaris eggs in biosolids was modified through a series of recovery efficiency experiments; we seeded soil samples with a known number of Ascaris suum eggs and assessed the effect of protocol modifications on egg recovery. We found the use of 1% 7X as a surfactant compared to 0.1% Tween 80 significantly improved recovery efficiency (two-sided t-test, t = 5.03, p = 0.007) while other protocol modifications-including different agitation and flotation methods-did not have a significant impact. Soil texture affected the egg recovery efficiency; sandy samples resulted in higher recovery compared to loamy samples processed using the same method (two-sided t-test, t = 2.56, p = 0.083). We documented a recovery efficiency of 73% for the final improved method using loamy soil in the lab. To field test the improved method, we processed soil samples from 100 households in Bangladesh and 100 households in Kenya from June to November 2015. The prevalence of any STH (Ascaris, Trichuris or hookworm) egg in soil was 78% in Bangladesh and 37% in Kenya. The median concentration of STH eggs in soil in positive samples was 0.59 eggs/g dry soil in Bangladesh and 0.15 eggs/g dry soil in Kenya. The prevalence of STH eggs in soil was significantly higher in Bangladesh than Kenya (chi-square, χ2 = 34.39, p < 0.001) as was the concentration (Mann-Whitney, z = 7.10, p < 0.001). This new method allows for detecting STH eggs in soil in low-resource settings and could be used for standardizing soil STH detection globally.
... The presence of helminthic ova, particularly Ascaris lumbricoides (the traditional reference parasite) along with its surrogate Ascaris suum, has been assayed in biosolids and wastewater effluent by a variety of methods, including microscopic examination and various PCR and quantitative PCR (qPCR) methods (20)(21)(22)(23). Because Ascaris is a ubiquitous parasite (infecting ϳ1.4 billion people worldwide) and has a direct life cycle that facilitates transmission by water, food, and person-to-person contact, it remains a logical reference pathogen for worm presence in sewage sludge (24,25). ...
Significant effort has gone into assessing the fate and removal of viruses, bacteria, and protozoan parasites during wastewater treatment to provide data addressing potential health risks associated with reuse options. Comparatively less is known about the fate of parasitic worm species ova in these complex systems. It is largely assumed that these helminths settle, are removed with the sludge, and consequently represent a relatively low risk for wastewater reuse applications. However, helminths are a highly diverse group of organisms that display a wide range of physical properties that complicate the application of a single treatment for helminth reduction during wastewater treatment. Moreover, their diverse biological and physical properties make some ova highly resistant to both disinfection (i.e., with chlorine or UV treatment) and physical removal (settling) through the wastewater treatment train, indicating that there may be reason to broaden the scope of our investigations into whether parasitic worm eggs can be identified in treated wastewater. The ubiquitous human parasitic nematode Enterobius vermicularis (pinworm) produces small, buoyant ova. Utilizing a novel diagnostic quantitative PCR (qPCR), this study monitored E. vermicularis presence at two full-scale wastewater treatment plants over the course of 8 months and demonstrated incomplete physical removal of E. vermicularis ova through tertiary treatment, with removal efficiencies approximating only 0.5 and 1.6 log10 at the two wastewater treatment plants based on qPCR. These findings demonstrate the need for more-diverse surrogates of helminthic ova to fully assess treatment performance with respect to reclaimed wastewaters.
... All eggs were counted. The 'split-spike technique' (Bowman et al., 2003) was used in determination of the accuracy of the method. ...
On-site sanitation systems such as dry pit, flush Pit, dual pit-latrines and septic tank are widely used around the globe. Due to the absence of any effective technology for the treatment of the septage generated from these sanitation systems, in most of the cases the contaminated septage is dumped in landfills or dumping yards without any treatment. There has been as such no effective development of Faecal Sludge treatment technology to ensure safe fate of the contaminated septage and in most of the occasion they have been found crudely disposed off in landfills or dumping yards and left untreated. The septage or its leachate has potential to cause serious health effects when drinking water gets contaminated. This extended abstract reports some of the results obtained when we employed black soldier fly larvae (BSFL) as a biological agent for the treatment and reduction of the amount of fresh human feces which forms a part of the septage. BSFL is a voracious eater of organic wastes and has been reported in literature to be very efficient agent for waste reduction and conversion into their body weight (Diener at al. 2011, Banks et al. 2014, Paz et al. 2015). Generally, the BSFL has a larval life of 14 days during which they consume food and gain weight before they turn into a prepupae wherein they avoid food. They turn into pupae and eventually, in a fly. Just like many other biological agents, these larvae are sensitive to environmental conditions such as temperature and humidity so that the most efficient waste reduction may take place at an optimum temperature and humidity. In this research our objective is to study the effect of the environmental parameters such as temperature and humidity on the substrate (human feces) reduction and weight gain by the BSFL. We performed a set of experiments involving BSFL put in fresh human feces under different environmental conditions. The temperatures were 25, 30 and 35 deg C which is within the prevailing temperature range (25-35 deg C) in most parts of India. For each temperature, relative humidity (RH) was varied between 55-85%. For each experiment, 50 numbers of 6 day old larvae were kept inside a box inside which 200 g of fresh human feces collected from a septic tank was spread at 2 cm thickness. Three replicates (human feces and BSFL) along with control (only contains human feces and no larvae) were made and each box was maintained at specific temperature and relative humidity inside an incubator. The BSFL as well as the substrate were weighed every other day till they kept on gaining weight. Figure 1.1 shows the Percentage weight gain of the BSFL and Percentage reduction in the substrate for the experiments run at 30 deg C and at different levels of relative humidity as an example. Due to brevity, we have included only the results obtained from the experiments run at Temperature,T=30 deg C and Relative Humidity (RH)= 55, 65, 75 and 85%.
The substrate reduction was found to be in the range of 3-17%, the highest being at RH of 65-75%. The weight gain of BSFL was observed to be in the range of 39-91%, the highest being at 85% RH. The results indicate that at 300C, the optimum utilization of food takes place at 65-75% RH. However, larval weight gain follows a different trend, monotonously increasing with respect to the relative humidity. The logical inference is that the larvae are most active in feeding as well as metabolising (catabolising) the substrate at RH of 65-75 %, while at higher RH the feeding rate is lower and the rate of metabolism (catabolism) is even lower. The weight gain of BSFL per unit substrate reduction for each larvae at RH=55, 65, 75 and 85% was calculated as 1.521, 0.983, 1.361 and 2.031 mg/gram. Hence, there is a net gain in weight of the BSFL at high humidity where, due to reduced activity, they cannot catabolize even the food they consume. As a result, there is a weight gain, which may at once look as anomalous result. The substrate consumed by each larvae was 0.613, 0.666, 0.656 and 0.579 g at RH=55, 65, 75 and 85%, respectively. This means that approximately 1500 numbers of BSFL may be required to consume 1 kg of human feces at 65-75% RH. Overall, BSFL showed a promising agent for the treatment of fecal sludge management.
... A Tabela 1 mostra a média e o desvio padrão das temperaturas máximas e mínimas, nas duas épocas do estudo, medidas a cada sete dias no interior da avaliação de ovos viáveis de helmintos, uma vez que a metodologia estabelecida pela legislação vigente (BRASIL, 2006;PARANÁ, 2009) refere-se a detecção, enumeração e determinação de viabilidade de ovos de Ascaris (BOWMAN; LITTLE; REIMERS, 2003;EPA, 2003). Na primavera, as análises laboratoriais de ovos viáveis de helmintos foram realizadas no Laboratório Senai CIC, seguindo a Resolução Conama 375/06 (Quadro 1). ...
O presente estudo teve por objetivo avaliar o comportamento de sólidos totais, pH e ovos viáveis de helmintos (Ascaris) em lodo de esgoto submetido a processo de revolvimento a cada sete dias por um período de 56 dias. O estudo foi desenvolvido em duas épocas, no outono e na primavera de 2010, com medições realizadas aos 0, 14, 28, 42 e 56 dias. Os resultados demonstram que ao longo do tempo houve um aumento dos sólidos totais e redução do pH do lodo de esgoto. Verificou-se uma redução significativa no número de ovos viáveis de helmintos (Ascaris) na medição de 56 dias nas duas épocas do estudo.
... However, current methodologies are not always effective for identification since experienced technicians are required, and therefore, results are often neither accurate nor reliable. Moreover, there is no universally accepted method to quantify them in sludge and biosolids [91]. The available analytical procedures commonly have two steps: (a) the separation of as many eggs as possible from other particles in the sample and (b) their visual identification under the microscope, where the concentrated sediment (pellet) contains many other types of particles. ...
... However, current methodologies are not always effective for identification since experienced technicians are required, and therefore, results are often neither accurate nor reliable. Moreover, there is no universally accepted method to quantify them in sludge and biosolids [91]. The available analytical procedures commonly have two steps: (a) the separation of as many eggs as possible from other particles in the sample and (b) their visual identification under the microscope, where the concentrated sediment (pellet) contains many other types of particles. ...
... Certain methods favour a coverslip placed over the top of the solution and centrifugation for the collection of ova, 7-10 while others filter out the ova. 11 The ova are subsequently viewed under a light microscope. The recovery rate of the large majority of these techniques is unknown; therefore, it is challenging to compare contamination levels between various sites or to assess the effect that a sanitation or hygiene intervention has on a community. ...
Background
Soil transmitted helminths (STH) continue to be associated with high burdens of disease, with an estimated 1.45 billion people infected with STH globally. The promotion and construction of latrines is considered the first barrier to prevent transmission of STH. The absence of a reliable method to extract STH ova from soil makes it challenging to examine whether the use of latrines may or may not have an effect on environmental contamination with ova. The present study evaluated the recovery rate of a method developed to extract STH ova from soil.
Methods
The adapted centrifugation and flotation technique was applied to 15 soil types, which were seeded with Ascaris suum ova. Soil type, soil moisture content, soil texture and organic matter content were assessed for each soil sample.
Results
The average ova recovery rate was 28.2%, with the recovery rate of the method decreasing with increasing soil moisture content, particle size and organic matter content. The association between recovery rate and organic matter content was statistically significant.
Conclusions
The present study identified a low recovery rate for an adapted centrifugation-flotation method, although this was similar to the recovery rate demonstrated by other methods developed for soil. Soil organic matter content was significantly associated with ova recovery rates.
... Source of hookworm ova for the study Experimental studies were performed using Ancylostoma caninum ova collected from different dog faecal samples (n = 6) after positive confirmation via microscopic examination. A. caninum ova were isolated from approximately 20 g of dog faecal samples using the flotation method described previously (Bowman et al. 2003). The isolated ova from six dog faecal samples were then mixed and assessed for viability using methyl blue vital stain. ...
Differentiation between viable and non-viable hookworm ova in environmental samples is necessary in order to identify hookworm contamination and implement strategies to mitigate re-infections in endemic regions. In this study, an untargeted metabolic profiling method was developed that utilised gas chromatography-mass spectrometry (GC-MS) in order to investigate hookworm ova viability. Ancylostoma caninum were used to investigate the metabolites within viable and non-viable ova. Univariate and multivariate statistical analysis of the data resulted in the identification of 53 significant metabolites across all hookworm ova samples. The major compounds observed in viable and non-viable hookworm ova were tetradecanoic acid, commonly known as myristic acid [fold change (FC) = 0.4], and dodecanoic acid, commonly known as lauric acid (FC = 0.388). Additionally, the viable ova had self-protecting metabolites such as prostaglandins, a typical feature absent in non-viable ova. The results of this study demonstrate that GC-MS methods can be used to determine the viability of canine hookworm ova. Further studies are needed to assess the applicability of GC-MS to detect viable hookworm ova in the mixed (viable and non-viable) populations from environmental samples and identify the metabolites specific to human hookworm species.
The probability of cysticercus bovis (CB) infection of cattle (cysticerci from Taenia saginata) in a country where T. saginata is not endemic (i.e. Australia) was assessed using a Quantitative Microbial Risk Assessment (QMRA) approach. Two important features of the QMRA were (i) a dose-response curve to describe ingestion of eggs of the helminth T. saginata (HE) by cattle and the development of cysticerci due to the infection, and (ii) characterisation of HE concentrations. Data limitations relating to HE quantification are described, and several other key variables provided the basis for a probabilistic QMRA model.
Data from over 554 sewage samples from 11 wastewater treatment plants (WWTPs) in Southern Australia indicated the background concentration of T. saginata eggs was low (<0.1 HE L-1 measured, 0.003 HE L-1 as an estimated baseline modelled on a ratio of Taenia:Ascaris determined from the literature). Such a low sewage concentration was estimated to require only a 2.2 log10 reduction value (LRV) via sewage treatment to maintain the baseline risk of CB equivalent to background levels in Australia. However, to protect against potential future detectable outbreaks of Taeniasis in the human population and all potential exposure scenarios considered, a 3.5 LRV for WWTP was considered appropriate with confirmation by appropriate sewage monitoring. In addition, analysis of several specific exposure scenarios using the QMRA indicated that LRV credits (0.5 to 2.0 LRV) could decrease the required LRV for wastewater treatment based on the size of the WWTP and on-site management strategies (e.g. restriction of recycled water use for livestock drinking water, the years of exposure for cattle to sites irrigated with recycled water, and the use of fodder off-site). Without such measures, a HE LRV of 4.0 is recommend for WWTPs to ensure adequate protection of systems with no on-site controls.
In dry areas, the need for irrigation to ensure agricultural production determines the use of all
available water sources. However, the water sources used for irrigation are often contaminated by
untreated or minimally treated wastewater. Microbial risks from reusing wastewater for vegetable
irrigation can be addressed by installing environmental barriers that pathogens must cross to reach
humans in the reuse system. Knowledge of pathogen flows inside the system and pathogen removal
potential is the first step towards devising a risk management strategy. This study assessed microbe
prevalence in farming systems in the Bolivian highlands that use wastewater-polluted sources for
irrigation of lettuce. Samples of soil, lettuce and different water sources used in the farming systems
were taken during one crop season and concentrations of coliphages, Escherichia coli and helminth
eggs were measured. The results showed high spread of these microorganisms throughout the
whole system. There was a significant correlation between microbial quality of water and of the
harvested produce for several microorganisms. The microbial prevalence in protected shallow wells
was found to be significantly lower than in other water sources. These findings can help formulate
feasible risk management strategies in contexts where conventional technologies for microbial
removal are not possible.
Accurate evaluation of viable Ascaris ova in wastewater is the key to mitigate Ascaris reinfections in endemic regions. In this study, the viability of Ascaris ova in raw wastewater was determined using three different detection methods: culture-based, BacLight Live/Dead staining and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR). Furthermore, comparative assessment of viability utilising the aforementioned detection methods was performed using seeded experiments in wastewater. The percentage of viability was; culture-based (82%), BacLight Live/Dead staining (87%) and PMA-qPCR (85%) respectively. Despite the fact that no statistical difference was shown in the viability determination among the three methods, PMA-qPCR based viability determination would be preferable over the other two methods for evaluating potential public health risks with A. suum ova due to its accuracy, least subjective and rapid reaction time.
Climate change, increase in population and scarcity of freshwater have led to a global demand for wastewater reuse in irrigation. However, wastewater has to be treated in order to minimize the presence of pathogens, in particular, the ova of soil-transmitted helminthes (STHs). Limiting the transmission via removal of STH ova, accurate assessment of risks and minimizing the exposure to the public have been recommended by health regulators. The World Health Organization (WHO) guideline specifies a limit of ≤1 ova/L for safe wastewater reuse. Additionally, the Australian Guidelines for Water recycling (AGWR) recommend a hydraulic retention time of over 25 days in a lagoon or stabilization pond to ensure a 4 log reduction value of helminth ova and to mitigate soil-transmitted helminths associated risks to humans. However, the lack of fast and sensitive methods for assessing the concentration of STH ova in wastewater poses a considerable challenge for an accurate risk assessment. Consequently, it has been difficult to control soil-transmitted helminthiasis despite effective mass drug administration. This limitation can be overcome with the advent of novel techniques for the detection of helminth ova. Therefore, this review presents an assessment of the current methods to detect the viable ova of soil-transmitted helminths in wastewater. Furthermore, the review focuses on the perspectives for the emerging state-of-the-art research and developments that have the potential to replace currently available conventional and polymerase chain reaction based methods and achieve the guidelines of the WHO in order to allow the safe reuse of wastewater for non-potable applications, thereby minimizing public health risks.
In dry areas, the need for irrigation to ensure agricultural production determines the use of all available water sources. However, the water sources used for irrigation are often contaminated by untreated or minimally treated wastewater. Microbial risks from reusing wastewater for vegetable irrigation can be addressed by installing environmental barriers that pathogens must cross to reach humans in the reuse system. Knowledge of pathogen flows inside the system and pathogen removal potential is the first step towards devising a risk management strategy. This study assessed microbe prevalence in farming systems in the Bolivian highlands that use wastewater-polluted sources for irrigation of lettuce. Samples of soil, lettuce and different water sources used in the farming systems were taken during one crop season and concentrations of coliphages, Escherichia coli and helminth eggs were measured. The results showed high spread of these microorganisms throughout the whole system. There was a significant correlation between microbial quality of water and of the harvested produce for several microorganisms. The microbial prevalence in protected shallow wells was found to be significantly lower than in other water sources. These findings can help formulate feasible risk management strategies in contexts where conventional technologies for microbial removal are not possible.
Objective
To review current evidence on infections related to the concentration of soil‐transmitted helminth (STH) eggs in wastewater, sludge and vegetables irrigated with wastewater or grown on sludge‐amended soils.
Method
Search of Web of Science, Science Direct, PubMed and Google Scholar databases for publications reporting on STH egg concentration in wastewater, sludge and vegetables and for epidemiological studies on wastewater/sludge reuse and STH infections.
Results
STH egg concentrations were variable but high in wastewater and sludge especially in developing countries. They ranged from 6 to 16,000 eggs/L in wastewater and from 0 to 23,000 eggs/g in sludge and far exceed limits set in the WHO guideline for wastewater/sludge reuse. Numbers of STH eggs on vegetables ranged from 0 to 100 eggs/g. The concentration of STH eggs in wastewater, sludge and vegetables therefore relates to risks of infection through different exposure routes.
Conclusion
Epidemiological evidence reveals an increased prevalence of STH infections associated with direct exposure to wastewater or sludge (farmers) and consumption of vegetables grown on soil treated with it. This calls for increased efforts to reduce the adverse health impact of wastewater and sludge reuse in line with the WHO multi‐barrier approach.
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Background and purpose: Sewage sludge can supply nutrients and improve soil condition. However, presence of microorganisms, particularly parasites could cause a variety of diseases in humans and animals. This study aimed at identifying parasites in the waste sludge of industrial sewage in Babolsar, north of Iran and comparing their concentration with the EPA standards.
Materials and methods: Sampling was conducted in the sludge sewage treatment plant in one industrial town from four sludge depot during six months. Nine samples were taken from each depot. In order to identify the parasites McMaster slide was used according to Bailenger method.
Results: This study showed that the average numbers of helminth eggs in pallets 1, 2, 3, and 4 were 4.42, 17.52, 7.65, and 7.3 per 4 grams dried sludge, respectively. The greatest numbers of helminth eggs, was associated with Toxocara and Hymenolepis diminuta.
Conclusion: In current investigation, the number of helminth eggs in the sludge was higher than that suggested by the EPA standards. The sludge studied was industrial, therefore, the possibility of parasitic infection caused by people is low. On the other hand, the numbers of Hymenolepis diminuta and Toxocara were high. As a result, the possibility of secondary pollution due to movement of animals is high.
Raw and partially treated wastewater has been widely used to maintain the global water demand. Presence of viable helminth ova and larvae in the wastewater raised significant public health concern especially when used for agriculture and aquaculture. Depending on the prevalence of helminth infections in communities, up to 1.0 × 103ova/larvae can be presented per litre of wastewater and 4 gm (dry weight) of sludge. Multi-barrier approaches including pathogen reduction, risk assessment, and exposure reduction have been suggested by health regulators to minimise the potential health risk. However, with a lack of a sensitive and specific method for the quantitative detection of viable helminth ova from wastewater, an accurate health risk assessment is difficult to achieve. As a result, helminth infections are difficult to control from the communities despite two decades of global effort (mass drug administration). Molecular methods can be more sensitive and specific than currently adapted culture-based and vital stain methods. The molecular methods, however, required more and thorough investigation for its ability with accurate quantification of viable helminth ova/larvae from wastewater and sludge samples. Understanding different cell stages and corresponding gene copy numbers is pivotal for accurate quantification of helminth ova/larvae in wastewater samples. Identifying specific genetic markers including protein, lipid, and metabolites using multiomics approach could be utilized for cheap, rapid, sensitive, specific and point of care detection tools for helminth ova and larva in the wastewater.
Soil-transmitted helminths (STHs) are a major health concern globally. Infection is mostly through contact with contaminated water, food or soil. Therefore to break the cycle of viable transmission STH eggs must be quantitatively detected in the environment. The effect of different reagents on the viability of Ascaris suum eggs during laboratory detection and quantification was assessed and different incubation solutions compared. Sulphuric acid gave a slightly higher recovery percentage of viable eggs (91.2%) than distilled water (90.0%) and 0.5% formalin (87.6%), although the difference was not statistically significant (p > 0.05). Acetoacetic acid, ethyl acetate, ammonium bicarbonate, zinc sulphate, magnesium sulphate and Tween 80, are reagents widely used in test protocols for the detection and quantification of STH eggs. Eggs were exposed to these reagents for different time durations. Acetoacetic acid resulted in the highest loss of viability (3.4 ±0.7% viable), while magnesium sulphate resulted in the least effect (88.5±1.2% viable). In conclusion the use of the selected reagents in the detection of these eggs was found to affect the viability of exposed eggs, especially during prolonged exposures. Therefore we recommended that eggs be exposed for ≤5 minutes, to reduce the risk of viability loss.
Polluted water can transmit lots of pathogens with various behaviors and resistances. Among the pathogens, the parasites are particularly important since they stand out for their high resistance to various environmental factors in addition to being associated to high morbidity and mortality rates in the developing countries, particularly children. The objective of this paper was to highlight the importance of health surveillance of parasites in water quality according to use and its relationship with the environment. The helminth eggs are the main health risk due to the safe use of wastewater or sludge in agriculture. Protozoan cysts like Cryptosporidium and Giardia, are difficult to remove from untreated drinking water due to its small size and resistance to commonly used oxidants such as chlorine. Although the routine monitoring of these cysts in water is not recommended, it is necessary to conduct research to detect its presence and to establish suitable guidelines according to our conditions.
At South East Water wastewater treatment plants (WwTPs) in Victoria, Australia, biosolids are stockpiled for three years in compliance with the State guidelines to achieve the highest pathogen reduction grade (T1), suitable for unrestricted use in agriculture and landscaping. However, extended stockpiling is costly, may increase odour nuisance and greenhouse gas emissions, and reduces the fertiliser value of the biosolids. A verification programme of sampling and analysis for enteric pathogens was conducted at two WwTPs where sludge is treated by aerobic and anaerobic digestion, air drying (in drying pans or solar drying sheds) and stockpiling, to enumerate and, if present, monitor the decay of a range of enteric pathogens and parasites. The sludge treatment processes at both WwTPs achieved T1 grade biosolids with respect to prescribed pathogenic bacterial numbers (<1 Salmonellaspp. 50 g⁻¹ dry solids (DS) and <100 Escherichia coli g⁻¹ DS) and >3 log10 enteric virus reduction after a storage period of one year. No Ascaris eggs were detected in the influent to the WwTPs, confirming previous studies that the presence of helminth infections in Victoria is extremely low and that Ascaris is not applicable as a control criterion for the microbiological quality of biosolids in the region.
Accurate quantitative measurement of viable hookworm ova from environmental samples is key to control the hookworm re-infections in endemic regions. In this study, the accuracy of three quantitative methods [culture-based, vital stain and Propidium Monoazide-quantitative PCR (PMA-qPCR)] were evaluated by enumerating 1,000 ± 50 Ancylostoma caninum ova in the laboratory. The culture-based method was able to quantify an average of 397 ± 59 viable hookworm ova. Similarly, vital stain and PMA-qPCR methods quantified 644 ± 87 and 587 ± 91 viable ova, respectively. The numbers of viable ova estimated by culture-based method were significantly ( P < 0.05) lower than vital stain and PMA-qPCR methods. Therefore, both PMA-qPCR and vital stains methods appear to be suitable for the quantitative detection of viable hookworm ova. However, PMA-qPCR would be preferable over vital stain method in scenarios where ova speciation is needed.
Because traditional methods used for sewage sludge parasitological examinations have low sensitivity, a new, highly effective method (own method – OM) was devised. The principle of this method is to eliminate the flocculent effect on the structure of sewage sludge by mechanically damaging floccules in the presence of surfactants and to increase the effectiveness of egg isolation processes in large volumes of liquids. The objective of this study was to estimate the effectiveness of the OM in detecting nematode eggs in sewage sludge samples containing flocculants. In the first stage, the effectiveness of the OM was compared to 4 other methods routinely used in parasitological examinations of dehydrated sewage sludge. Next, method standardisation was performed using sewage sludge samples supplemented with eggs from 3 parasite species (Ascaris suum, Toxocara canis and Trichuris vulpis).
Frequent monitoring of anaerobic digestion processes for pathogen destruction is both cost and time prohibitive. The use of surrogates to supplement regulatory monitoring may be one solution. To evaluate surrogates, a semi-batch bench-scale anaerobic digester design was tested. Bench-scale reactors were operated under mesophilic (36 °C) and thermophilic (53-55 °C) conditions, with a 15 day solids retention time. Biosolids from different facilities and during different seasons were examined. USEPA regulated pathogens and surrogate organisms were enumerated at different times throughout each experiment. The surrogate organisms included fecal coliforms, E. coli, enterococci, male-specific and somatic coliphages, Clostridium perfringens, and bacterial spores. Male-specific coliphages tested well as a potential surrogate organism for virus inactivation. None of the tested surrogate organisms correlated well with helminth inactivation under the conditions studied. There were statistically significant differences in the inactivation rates between the facilities in this study, but not between seasons.
In Mexico, physicochemical sludge contains high levels of pathogens; and alkaline stabilization is an alternative for their control. However, the odours caused mainly by ammonia generation represent a disadvantage. On the other hand, the ammonia is known as an effective disinfectant. The aim of this study was to evaluate the disinfectant properties of ammonia in sludge, and use it in a closed alkaline stabilization system, which, not only copes with odours but also increases the efficiency of the process. Raw sludge from a municipal wastewater treatment plant using a physicochemical process was used. Ammonia was applied in doses from 10 to 50% w/w; also, doses from 5 to 40% of CaO were applied in open and closed systems and raw and treated sludge quality was evaluated. Results showed that ammonia removed 6 and 5 logs of faecal coliforms and Salmonella spp., respectively and up to 94% of viable helminth ova. The closed system was more efficient than the open system when applying doses from 5 to 20% of CaO. Finally, the results indicate that the ammonia represents an alternative to disinfecting wastewater sludge and it can be used to enhance alkaline stabilization processes.
Regulations for land application of wastewater sludges require the performing of treatment with a high efficiency on pathogens reduction. A reduction of 61% of the Volatile Solids content of the sludge, a reduction of fecal coliform, polio virus and Ascaris egg of 5.5, 4.0 and 2.6 respectively are achieved with a thermophilic/mesophilic Two Phase Anaerobic Digestion process. According to the EPA 40 CFR 503 regulation, the process produces Class A biosolids and could be recommended as a Process to Further Reduce Pathogens.
The objectives of the research grant were to: assess the presence and densities of resistant stages of parasites in municipal wastewater sludges (sewage) in northern United States; compare the results of the study with the results of a previous study of sludges in southern United States; to evaluate several decontamination techniques for their effectiveness in inactivating parasites in waste sludges; and develop a standard method for the parasitologic examination of waste sludges. Sludge samples from all phases of treatment (i.e., primary, etc.) were collected during the fall, winter and summer from 48 municipal wastewater treatment plants located in New York, Ohio, Minnesota, and Washington.
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A field investigation of ascaris ova survival in domestic sludge applied to land
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The two phase anaerobic digestion process
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