THE EFFECTS OF TECHNICAL STEPS USED IN EXISTING SANITATION HELMINTH TEST METHODS ON ASCARIS SUUM EGG RECOVERY FROM PIG FECES

No helminth recovery method is universally accepted for processing sanitation and soil samples (Gyawali, 2017). Existing methods entail the following basic principles: washing of a sample (using a solution that can dissociate bonds between helminth eggs and the sample matrix) and subsequent filtration over sieves, flotation (using density gradients to separate eggs from other particles), phase extraction to remove protein and lipids, microscopic analysis of the resulting pellet to quantify eggs, and incubation for viability assessment (Rocha et al., 2016; Ravindran et al., 2019). Furthermore, many technical aspects that are related to these steps can impact how well the method can recover eggs, including centrifugation speeds and times, choice of wash and flotation solutions, and specific gravity (sp. gr.) of the latter (Collender et al., 2015; Amoah et al., 2017).

Common sanitation helminth methods currently used globally include the United States Environmental Protection Agency (USEPA) method, the Mexican standard for wastewater analysis, the Bailenger method, and the Pollution Research Group (PRG) helminth method. The last was established by our group, implemented in our laboratory for helminth testing, and forms the baseline for this study (Ayres and Mara, 1996; USEPA, 2003; Secretaría de Economía, 2012; Velkushanova et al., 2021). Table I outlines the technical steps for each of these methods and forms the basis upon which all experiments in this study were designed.

Homogenization of a sample enhances consistent egg recovery before processing, as well as after soaking in the wash solution, the latter of which ensures the dissociation of eggs from the matrix (Bowman et al., 2003). For sludge samples, it is recommended that a surfactant be used, e.g., 7X®, Triton® X-100, Tween® 20, or Tween 80 (Collender et al., 2015; Velkushanova et al., 2021). Some surfactants possess antimicrobial properties, thus preventing excessive biological contamination (Amoah et al., 2017). Soil type and texture of samples can affect the recovery of eggs, but soaking and homogenization of soil samples in ammonium bicarbonate allows for the dissociation of bonds that form between the surface layer of the eggshell and silica particles in soil (Nunes et al., 1994; Hawksworth et al., 2010).

Often the use of sieves and respective mesh or pore sizes are looked at to determine the efficacy of egg recovery relative to the size of eggs of various helminth species (Amoah et al., 2017; Ravindran et al., 2019). No studies, however, investigated how the mode of washing over these sieves affects the separation of eggs from the sample matrix. The USEPA method recommends that samples be washed over the sieve using a spray bottle; thus no pressure is applied to push eggs released from the sample through the top sieve (USEPA, 2003). The Mexican standard for wastewater analysis uses an exact, measured amount of water to wash the sample over the sieve, most likely from a measuring cylinder, thus without pressure (Secretaría de Economía, 2012). Furthermore, both methods recommend that the first washing be done on a sieve placed over a beaker such that the wash water can be collected. This could allow egg loss over the sides of the sieve if it does not fit exactly over the beaker. The PRG helminth method uses a 100 µm sieve over a 20 µm sieve for all helminth eggs, and samples are washed under pressure using a hose or shower head attached to the tap such that eggs are pushed through the top sieve and are contained in the retentate on the 20 µm sieve (Velkushanova et al., 2021).

Comparative data are lacking in terms of the best wash solution for specific sample types and optimal centrifugation speeds and times for egg retrieval (Amoah et al., 2017; Ravindran et al., 2019). Centrifugation after washing is essential for effective sedimentation of eggs in the filtrate, such that eggs are not lost in the supernatant (Ravindran et al., 2019). Many methods recommend gravitational sedimentation of the sample after washing, followed by vacuum aspiration of the supernatant (Ayres and Mara, 1996; USEPA, 2003), after which the sediment is transferred to test tubes and centrifuged for up to 1,000 g (Bowman et al., 2003).

Flotation solutions are used to create a difference in density between eggs, other particles in the sample, and the suspension medium, such that eggs can be separated from residual matter that was not removed during the washing and sieving step (Dryden et al., 2005; Amoah et al., 2017). The flotation solution needs to be denser than the eggs with a minimum sp. gr. of 1.25 and is made up (using a hydrometer) of a sp. gr. that allows all eggs to float (Dryden et al., 2005); thus eggs that are lighter than the other particulate matter and the flotation solution are buoyant (David and Lindquist, 1982; Ravindran et al., 2019). Once centrifuged, the particulate matter packs tightly into a pellet at the bottom of the tube, eggs float up toward the surface of the flotation medium, and the supernatant is collected. According to David and Lindquist (1982), the relative density of soil-transmitted helminth eggs ranges from 1.05 to 1.23, with Ascaris suum eggs having a relative density of 1.13.

Commonly used flotation solutions include zinc sulfate and magnesium sulfate, at sp. gr. of 1.18, 1.2, and 1.3 (Ayres and Mara, 1996; USEPA, 2003; Secretaría de Economía, 2012; Velkushanova et al., 2021).

The present study is the second in a series of 3 and was designed around recommendations made by (Naidoo and Archer, 2024a), which looked at the effects of all reagents and chemicals used in existing helminth test methods on the viability of A. suum eggs. The optimum chemicals used for testing the technical steps explored in this study were selected based on the first study results, i.e., the best flotation solution relative to sp. gr. and best wash solutions for different sample types. Other technical aspects investigated here include the mode of washing and optimal centrifugation speeds and times after washing and after flotation, all for the highest egg recovery. Data from this study will be used for the design of a final helminth test method to use on a variety of sanitation and environmental samples.

Ascaris suum eggs were isolated from the feces of research pigs (PRG helminth method). Egg stocks were made up in 0.5% formalin at approximately 200 eggs per milliliter. A slurry of helminth-negative pig feces was made up by blending the feces with water to facilitate efficient spiking and mixing of eggs, as pig feces alone are dry. Types of environmental or sanitation samples for testing in the final experiment were selected as being the most common types received by water and sanitation laboratories. Spiking was then performed with 1 ml egg stock per 10 g slurry samples, and 10 g test sample, for each experiment below. Every comparison factor in each experiment was performed using 5 replicates.

The mode of washing used, i.e., using pressure from a tap with a hose or shower nozzle fitted and without pressure using a wash bottle, was tested against egg recovery. Spiked samples, prepared as above, were either washed using tap water under pressure or with no pressure, onto a set of sieves (200 mm diameter and 50 mm deep, always with the 100 µm sieve on top of the 20 µm). Retentate on the 20 µm sieve was collected into 15 ml graduated, conical, plastic test tubes (Falcon tubes) and centrifuged at 1,050 and 1,512 g for 5, 10, and 15 min.

Since we were testing the efficacy of the mode of washing and centrifugation speeds and times only, to avoid confounding results, we could not use a wash solution on the samples before washing them over the sieves, nor perform the flotation step to complete the processing part of the method before microscopic analysis. After centrifugation, all supernatants were discarded. Pellets were topped up with water to 5 ml and dislodged using a vortex to homogenize them, and 1 ml was removed for immediate analysis. Egg recovery was then extrapolated to 5 ml. This was to establish an estimated egg recovery without any possible egg loss during the normal subsequent flotation step. The remaining 4 ml was stored at 4 C for full processing later once the flotation step had been optimized.

After determining the optimal flotation solution, sp. gr., centrifugation speeds, and times, pellets from the 4 ml samples were floated using zinc sulfate as per the flotation experiment below and analyzed for egg recovery. This figure was added to the recovery from the 1 ml sample to establish total egg recovery.

Zinc sulfate, magnesium sulfate, and sodium nitrate were tested for optimal egg recovery, each at sp. gr. of 1.18, 1.2, and 1.3. Centrifugation speeds and times for flotation were tested against egg recovery, i.e., 672 and 1,050 g for 5, 10, and 15 min. Spiked samples were washed under pressurized tap water over the set of sieves. Retentate on the 20 µm sieve was collected into 15 ml Falcon tubes and centrifuged at 1,512 g for 10 min (the optimum mode of washing and centrifugation speed and time were selected based on the previous experiment). The supernatant was discarded, and the pellet dislodged with an applicator stick whilst simultaneously pipetting 3 ml at a time of flotation solution into each tube and using a vortex mixer to ensure homogenization. The sample was topped up to 14 ml with a flotation solution and centrifuged. The supernatant was then poured onto a 20 µm sieve (100 mm diameter, 50 mm deep) and rinsed with tap water. The retentate was pipetted into 15 ml Falcon tubes and centrifuged at 1,512 g for 10 min. The supernatant fluid was discarded, and the pellet was analyzed for egg recovery using light microscopy.

Two different wash solutions plus water as a control were used on various types of samples to determine which solution resulted in the best egg recovery. Sample types included water, effluent, ventilated improved pit latrine (VIP), urine diversion dry toilet (UDDT), dried (soaked for both 4 and 24 hr), fatty, and septic tank sludges, and soil and were sub-sampled and spiked with a known number of A. suum eggs before testing began. The selection of wash solutions was based on data concerning their effect on A. suum egg viability and ability to clean up the sample as tested for by Naidoo and Archer, 2024a. The preferred wash solutions determined in the first study were ammonium bicarbonate (119 g/L) and 0.1% 7X, and water was included as a control. All experiments were performed in quintuplicate (Table II).

Samples were then washed over a set of sieves under pressurized tap water. Retentate on the 20 µm sieve was collected into as many 15 ml Falcon tubes as was required, depending on sample type. The tubes were centrifuged at 1,512 g for 10 min, supernatants were discarded, and pellets floated with zinc sulfate (sp. gr. 1.3), processed, and analyzed using light microscopy. The final pellets of the fatty sludge samples were large and required an extraction step, but eggs were being lost in this process (see Naidoo and Archer, 2024b). Fatty sludge samples were re-run a few times until we found an effective way of counting eggs in the final pellet. Eventually, the resulting pellets were resuspended to 1 ml with water in the test tubes and homogenized. The drops were counted, and half that number were analyzed. The number of eggs counted was doubled to calculate total egg recovery.

When looking at the effect of washing mode and centrifugation time alone, significant effects on egg recovery were noted (P < 0.001), but centrifugation speed alone was insignificant (P = 0.540). The combination of washing mode and centrifugation speed did not impact egg recovery (P = 0.630), but when nested with centrifugation time, egg recovery was significantly affected (P < 0.001). Figures 1 and 2 indicate that samples need to be washed under pressure. This physically separates larger particles on the 100 µm sieve from eggs, whilst also pushing eggs through the 100 µm mesh of the top sieve onto the 20 µm mesh sieve below. Furthermore, the back of a gloved hand can be used to mechanically break up clumps and gently push eggs and smaller particulate matter through the top sieve. Using a wash bottle to wash the sample on the 100 µm sieve made it difficult to break up larger particles and push eggs through the sieve pores with the finer debris. The sample never quite looked adequately washed, whereas with pressure, the water eventually ran clear from the 100 µm sieve, indicating that washing was complete.

The extrapolated figures, when the flotation step was not included (Fig. 1), indicated that both times of 10 and 15 min at a speed of 1,050 g resulted in >90% egg recovery. Recovery, after including the flotation step (Fig. 2), indicated that centrifugation at 1,512 g produced a higher egg recovery than at 1,050 g, for 10 min, but when centrifugation time was increased to 15 min, there was no difference in the percentage of eggs recovered at both speeds. It was, however, noted that the pellet compacted better at 1,512 g, making it easier to discard the supernatant without losing eggs before performing the flotation step.

Flotation solution alone played a significant role in egg retrieval (P < 0.001), and across all 3 solutions, egg recovery increased as the sp. gr. was increased (Figs. 3, 4; P < 0.001). Furthermore, a significant effect was seen when recovery was nested with centrifugation speed alone (P < 0.001) and speed and time together (P < 0.001). Egg recovery at sp. gr. 1.18 was extremely low for all 3 flotation solutions, indicating that A. suum eggs require a denser solution for better separation from particles (sp. gr. alone on egg recovery: P < 0.001).

The nested effect of specific gravity, centrifugation speed, and centrifugation time was also significant in terms of how well eggs were recovered (P < 0.001). At sp. gr. 1.2, egg recovery was still very low, with all 3 solutions recovering <50% of the spiked eggs. This indicated that the density of solutions plays a more important role in egg recovery than centrifugation speeds and times. Even when samples are spun down for a longer period (Figs. 3, 4), separation from particles was not completely successful at lower specific gravities.

Zinc sulfate resulted in the best egg recovery at sp. gr. 1.3 when centrifuged at 672 g for 15 min (> 90%). It was therefore selected as the ideal flotation solution. It was also noted that eggs required a slower speed and longer centrifugation time to separate from particulate matter and successfully float up the supernatant column. At sp. gr. 1.3, both centrifugation speed and time made a difference in egg recovery. Eggs require a slower speed (effects of speed alone on egg retrieval: P < 0.001) and longer time (effects of time alone on egg retrieval: P < 0.001) to separate from denser particles and float up the supernatant column when being centrifuged. Egg recovery with sodium nitrate was very low (< 40%) across all densities, speeds, and times, and was therefore deemed unfit as a flotation solution (Fig. 5). Magnesium sulfate was successful at sp. gr. 1.3, and centrifugation at 672 g for 15 min; however, recovery remained at < 90% (Fig. 4).

When looking at the effect of wash solution alone on egg recovery, no significant effect was seen; however, the interactive effect with sample type was significant for egg recovery (P = 0.672 and P = 0.025). Across all 3 wash solutions, egg recovery was ± 90% for each sample type, except in the case of fatty sludges, which most likely accounts for the significant interactive effect of the ANOVA. The major difference was not necessarily observed in egg recovery values, but instead when processing and analyzing the different sample types. For water and effluent samples, washing with water was sufficient; however, if samples had to be incubated for viability assessment, washing with a chemical solution assisted better in preventing an increase in microbial contamination.

For VIP and septic tank sludge, 7X was the most successful in breaking down the sample and reducing the final pellet size, as well as making microscopy the easiest as the pellet dissociated well, resulting in egg recovery of >91%. Dried sludge was soaked for 4 and 24 hr, and both resulted in similar egg viability across solutions, indicating that samples can be soaked overnight in either 7X or ammonium bicarbonate. When comparing all 3 wash solutions, fatty sludge was best broken down by 7X. These samples were particularly difficult to handle and process, resulting in fatty deposits even after washing with a surfactant. The final pellets for microscopy were large; thus only half the pellet was analyzed, and the values were extrapolated. Egg recovery was highest for UDDT sludge and soil when washed with ammonium bicarbonate. The final pellets were also small enough to microscopically analyze with ease.

Nunes et al. (1994) noted that a protocol specific to sample type is important for the successful retrieval of helminth eggs from samples. This was exactly our concern and led to our extensive study (written as a series of 3 studies) of which this is number 2.

The USEPA method uses a spray bottle to wash samples over the sieve (USEPA, 2003), which could result in residual egg loss or eggs being trapped in the pores of the mesh on the top sieve. The same can be said for the Mexican standard for wastewater analysis, which recommends that a measured amount be poured over the sample on the sieves (Secretaría de Economía, 2012). Our data indicated that washing under pressure recovers far more eggs than the alternative.

Actual recovery data from washing (and after flotation was performed) indicated that centrifugation at 1,512 g performed best for egg recovery. Since both centrifugation times of 10 and 15 min were successful, we recommend the former, as it means exerting less pressure on the eggs. Manser et al. (2016) reported that egg recovery increased with increasing centrifugation speed and time. Eggs may remain in suspension for prolonged periods, thus longer centrifugation speeds resulted in better egg retrieval; however, excess debris interfered with microscopic analysis when eggs were centrifuged at 2,058 g for 10 min. No visible degradation of the eggs was evident at 1,512 g for 10 min (Manser et al., 2016), which corroborates our study and justifies the recommended speed and time.

Santarem et al. (2009) reported no difference in egg recovery between sp. gr. of 1.2, 1.25, and 1.3 across solutions, but zinc sulfate and sodium nitrate performed much better than magnesium sulfate. They also stated that sodium nitrate formed crystals that subsequently hindered microscopic analysis. We found that magnesium sulfate precipitated out of solution and formed crystals at the bottom of the bottle when stored and is therefore deemed unfit as a flotation solution. We also found that sodium nitrate recovered <40% of spiked eggs, thus making it unsuitable as a flotation solution. Smith (1991) reported that prolonged exposure to magnesium sulfate is toxic to eggs, thus supporting its exclusion.

Sá et al. (2017) compared the flotation efficacy of zinc sulfate at sp. gr. 1.3 without centrifugation, and at sp. gr. 1.35 and speed of 2,058 g for 5 min. They found that centrifugation resulted in 57% more eggs recovered than in passive flotation. This also indicates that the higher the sp. gr., the better the egg retrieval, and this also corroborates our findings. It should be noted, however, that physicochemical properties, such as the viscosity of the solution, affect egg recovery, and the denser the solution, the higher the viscosity (Oge and Oge, 2000). This would create resistance as eggs float up the liquid column and thus impede recovery.

We found that A. suum eggs required a denser solution than sp. gr. 1.18 to float, which was also observed by Quinn et al. (1980) and Nunes et al. (1994). Souza et al. (2011) reported 36% Ascaris egg recovery when eggs were spiked into wastewater samples and then floated with zinc sulfate at sp. gr. 1.18, also supporting our study’s findings. Quinn et al. (1980) found that recovery using zinc sulfate at sp. gr. 1.2 was far lower than magnesium sulfate at sp. gr. 1.27. We, however, found that not only does sp. gr. impact egg recovery but so does the flotation solution, with zinc sulfate being the optimum solution at sp. gr. 1.3.

Bowman et al (2003) reported that the choice of wash solution affects the dissociation of eggs from the sample and that 7X was superior to Triton X-100 and Tween 80 for egg recovery. Figure 6 indicates that 7X recovered the most eggs for VIP and septic tank sludge, and it is therefore recommended for these sample types. It is also recommended for fatty sludge samples; however, egg recovery was not as efficient. Manser et al. (2016) reported that helminth eggs can be trapped by fats in a sludge sample. In this study, eggs were lost during sample processing, most likely due to the wash solution not being able to fully dissociate eggs from lipids in fatty sludge.

Ammonium bicarbonate facilitates the dissociation of bonds formed between eggs and silica particles, and UDDT sludge is known to contain soil (Hawksworth et al., 2010); thus final pellets were easily dislodged, and small particles well dispersed for easier microscopy. It is therefore recommended for use on UDDT and soil samples. Eggs can adhere to a variety of components in such sludges, such as humic and fulvic acids, that are commonly found in soil (Rocha et al., 2016). Ammonium bicarbonate would therefore facilitate the dissociation of these bonds and allow the separation of silica particles from eggs, with Hawksworth et al. (2010) reporting 77.28% egg recovery from spiked UDDT sludge samples.

To date, no single method exists that is simple, time- and cost-efficient for all sample types and helminth species (Gyawali, 2017). Analysis of the technical steps was imperative for the development of such a method.

To ensure the dissociation of any bonds formed between helminth eggs and particulate matter, samples must be homogenized in a wash solution before processing. We conclude that water and liquid samples can be processed either without the use of a wash solution or with 7X. For sludge samples, we recommend the use of 7X as a surfactant to break down lipids. For soil or soil-containing samples, ammonium bicarbonate should be used for bond dissociation. Dried sludge can be soaked for a minimum of 4 hr and up to 24 hr if necessary in either 7X or ammonium bicarbonate. The mode of washing is important. Water under pressure is required when washing samples over the sieves to facilitate the breaking up of the sample matrix and pushing eggs through the coarser mesh of the top sieve. Samples should then be centrifuged at 1,512 g for 10 min for optimal egg recovery and a well-compacted pellet at the bottom of the test tube to allow for the supernatant to be easily discarded before floating. Zinc sulfate at sp. gr. 1.3 and centrifugation at 672 g for 15 min recovered the highest percentage of spiked eggs and is therefore recommended for flotation. Based on the data collected, the final method at the end of this study recovered >90% of eggs in most sample types, except fatty sludge.

Future research includes further testing of steps that could be included in the protocol to enhance the recovery of helminth eggs from fatty sludge. Testing the efficacy of the phase extraction step for egg recovery must also be undertaken to conclude the testing of every procedure in conventional sanitation and environmental helminth test methods (study 3 of our series does this).

We would like to thank the Water Research Commission (WRC) of South Africa and the National Research Foundation (NRF) for funding this project, as well as Merissa Naidoo for her invaluable technical assistance and input. Ethical approval for this study was granted by both the Animal Research Ethics Committee (AREC) and the Biomedical Research Ethics Committee (BREC) of the University of KwaZulu-Natal (AREC/071/018 and BREC/00002794/2021).