ASCARIS SUUM EGG RECOVERY FROM SLUDGE SAMPLES AFTER PHASE EXTRACTION

Typical helminth test methods used for sanitation and environmental samples comprise the same basic steps: washing the sample over a set of sieves (filtration), flotation using density gradients for separation of eggs from heavier particles, phase extraction to further remove debris, microscopic analysis, and incubation of the eggs to determine viability (USEPA, 2003; Rocha et al., 2016; Amoah et al., 2017; Pakdad et al., 2018; Ravindran et al., 2019). Two of these steps, flotation and phase extraction, were derived from stand-alone parasitology methods for the concentration of parasites from individual fecal samples (Cheeseborough, 1981).

Phase extraction involves using a hydrophilic solution and a lipophilic solvent to separate lipids and proteins from the test sample, thus reducing the final pellet size for microscopic analysis (Nelson and Darby, 2001; Collender et al., 2015; Rocha et al., 2016). Specific volumes of each chemical are added to a sample in a test tube that is vigorously shaken and centrifuged at 1,512 g. The sample separates into 2 phases, light and heavy, with a plug of organic material/debris that forms between. After centrifugation, helminth eggs are supposedly concentrated in the sedimented deposit, and the entire supernatant (that includes both phases and the middle disc) is discarded (Nelson and Darby, 2001; Collender et al., 2015; Rocha et al., 2016; Amoah et al., 2017).

Allen and Ridley (1970) originally presented ‘the extraction step’ as a pathology laboratory method, the formal–ether concentration, for isolating intestinal parasites from individual stool samples using a combination of 10% formalin and diethyl ether. Acetoacetic buffer, formalin, or acid alcohol (mixture of sulfuric acid and ethanol) (Ayres and Mara, 1996; Nelson and Darby, 2001; Secretaría de Economía, 2012; Velkushanova et al., 2021) are commonly used in sanitation methods to form the hydrophilic phase, whereas ethyl acetate or diethyl ether form the lipophilic phase (Rude et al., 1987; USEPA, 1999; Nelson and Darby, 2001; Rocha et al., 2016; Amoah et al., 2017). Some methods recommend that extraction be performed before the flotation step (Bailenger method), whereas others recommend that, if the pellet is large, it be performed afterward (PRG Helminth Method, old U.S. Environmental Protection Agency [USEPA] Method and the Mexican Standard for Wastewater Analysis) (Satchwell, 1986; Ayres and Mara, 1996; U.S. EPA, 1999; Secretaría de Economía, 2012; Velkushanova et al., 2021).

Some studies indicated that extraction solutions negatively affect egg viability (Nelson and Darby, 2001; Rocha et al., 2016) and recommend that this step not be included in sample processing; however, if included, then exposure time should be minimal (Nelson and Darby, 2001). It has been reported that although organic matter is removed upon extraction, it can result in the loss of approximately 95% of eggs because of their distortion (Satchwell, 1986). Nelson and Darby (2001) reported that eggs were lost after extraction, either by destruction due to the activity of the solvent or when discarding the layers above the pellet. The original USEPA method (1999) recommended that extraction be performed on samples; however, the updated version (USEPA, 2003) excludes the extraction step. Gaspard et al. (1995) used ethyl acetate to emulsify the sample and break down organic material after the addition of a detergent but before the flotation step. The PRG helminth method suggests extraction after flotation with 10% formalin and diethyl ether or ethyl acetate, only when the pellet is very large and impedes microscopic analysis.

In this study we examined the single and combination effects of various extraction solutions used in existing methods on the viability of Ascaris suum eggs. The present study therefore aimed at determining egg recoverability from samples that were subjected to a phase extraction.

All extraction chemical combinations were used to test water, primary sludge, and fatty sludge in replicates of five for the efficacy of egg recovery. Water acted as a control, and primary and fatty sludges were chosen as commonly tested sanitation samples. Although primary sludge (pretreated, predigested inlet sludge from a wastewater treatment plant) is not too problematic to process and examine microscopically, fatty sludge (upper scum layer and some sludge from the inlet chamber of an anaerobic baffled reactor) is the most difficult sample type to process and results in a large final pellet. Both animal and biomedical research ethics approvals were obtained from the University of KwaZulu-Natal (AREC/071/018 and BREC/00002794/2021).

Ascaris suum eggs were isolated from the feces of research pigs. Approximately 250 eggs were added to 10 g of primary sludge, 10 g of fatty sludge, and 14 ml of water in plastic graduated conical test tubes and these were thoroughly homogenized. For the sludge samples, 0.1% of 7X (a laboratory surfactant manufactured by MP Biomedicals [Irvine, California] used to dissolve fats in sludge samples) was added and the samples were mixed well. Sample processing was done according to existing standard helminth egg recovery protocols. Each sample was then washed using tap water under pressure over a set of pan sieves (each 200 mm in diameter and 50 mm deep, with the 100-μm-mesh sieve placed on top of the 20-μm-mesh sieve). The retentate on the 20-μm sieve was collected into 2 × 15-ml test tubes. The tubes were centrifuged at 1,512 g for 10 min, the supernatants were discarded, and deposits floated with zinc sulfate of specific gravity (sp. gr.) 1.3 using centrifugation at 672 g for 15 min. The supernatant was poured onto a 100-mm-diameter 20-μm-mesh sieve and thoroughly rinsed with water, after which the retentate was collected into a 15-ml test tube and centrifuged at 1,512 g for 10 min. The water samples were also centrifuged.

After the final centrifugation step, all supernatants were discarded and extraction solutions were added in the following combinations for each, using 7 ml of buffer + 4 ml of solvent: 10% formalin + ethyl acetate, 10% formalin + diethyl ether, acetoacetic buffer + ethyl acetate, acetoacetic buffer + diethyl ether, and acid alcohol + ethyl acetate. The tubes were vigorously shaken for 1 min to ensure complete dislodgement of the pellets and then centrifuged at 1,512 g for 10 min. A disc of organic material (debris) formed between the buffer and solvent layers (Fig. 1). The supernatant (including the 2 phases and the interphase disc) would normally be discarded, but instead it was removed with a 3-ml plastic Pasteur pipette and expelled onto a 20-μm sieve and rinsed thoroughly to remove all chemicals. The retentate remaining on the sieve was collected into a clean test tube and centrifuged at 1,512 g for 10 min. The entire pellet that remained in the original test tube was immediately analyzed for egg recovery using a light microscope. This was done by loosening the pellet with a pipette and adding a few drops of water to facilitate suction into a pipette and full transfer onto a glass slide. The washed and centrifuged supernatant portion was also microscopically analyzed for egg recovery, and the sum of both analyses gave the total egg recovery.

Pellet recovery (normally analyzed) refers to the percentage of added eggs recovered and counted in the pellet after the phase extraction contents were pipetted off. Supernatant recovery (not normally analyzed because it is discarded) refers to the percentage of added eggs recovered in both phases plus the disc of organic matter in between (Fig. 1). Total egg recovery refers to the sum of eggs counted in the pellet and in the supernatant and should be equal to what was added (±90%, being within 10% of eggs added, the criterion set for successful egg recovery for this study).

The true efficacy of the phase extraction is seen in the eggs recovered from the pellet only from each of the chemical combinations. Egg recovery from the different extraction combinations alone was significant (P < 0.001). Sample type alone also significantly affected egg recovery after extraction (P < 0.001). The interactive effects of extraction combination and sample type also significantly affected egg recovery (P < 0.001), indicating that both the ability of the extraction solutions to separate eggs from organic material (including their respective synergistic effects) and the sample matrix play a role in egg recovery. Table I shows egg recovery in both the pellet and supernatant and total egg recovery for each extraction combination per sample type. The mean total egg recovery (the sum of eggs in the pellet and supernatant) in water, primary sludge, and fatty sludge was 95.1, 90.3, and 70.1%, respectively.

Table I shows data from phase extractions conducted in water samples. Although total egg recovery across the 5 combinations was high (>85%), the actual egg recovery for formalin + ethyl acetate, formalin + diethyl ether, acetoacetic buffer + ethyl acetate, and acetoacetic buffer + diethyl ether was very low, at 8.2, 13.7, 0.8, and 0.5% respectively. The acid alcohol + ethyl acetate combination, however, showed promising results, with a pellet egg recovery of 93.2%. Extractions performed on primary sludge samples produced slightly better results; however, egg recovery was still far too low (22.0–46.1%) for the first 4 combinations. The acid alcohol + ethyl acetate pairing once again resulted in sufficient egg recovery of 89.8%; however, this combination did not remove much organic matter. Visually, the final pellet size was not reduced, making the extraction step fruitless. More eggs were recovered after extracting with formalin (48.7% and 32.6%) than with acetoacetic buffer (1.3% and 0.4%) and acid alcohol + ethyl acetate (57.3%), but the final pellet size was not reduced. Generally, total egg recovery was much lower in fatty sludge samples, which were more difficult to process and analyze microscopically. Across the first 4 extraction combinations and 3 sample types, eggs did end up in the supernatant, i.e., floating in the hydrophilic phase, trapped in the disc, or above the disc in the solvent (Fig. 1). We then removed the solvent separately from the disc using a pipette and did not find eggs present. It was therefore concluded that the eggs were trapped with the organic material in the disc. The acid-alcohol flotation was the most successful in terms of egg recovery across sample types but was unable to reduce the pellet size, making it ineffective.

Extraction is generally effective in the removal of organic material and reduction of pellet size; however, the reagents have toxic effects on the viability of the eggs (Rocha et al., 2016). One of the steps involved in sludge processing to recover helminth eggs is flotation. This involves the use of density gradients to separate eggs from particulate matter, where the solution must be denser than the eggs to allow for them to float up the supernatant column (Rocha et al., 2016). The same principle can be used to explain the differences in egg recovery across different extraction solutions. The sp. gr. of Ascaris eggs is 1.13 (David and Lindquist, 1982). Table II shows the sp. gr. of all 5 tested extraction solutions, and clearly, Ascaris eggs are denser than all solutions. This would mean that eggs should settle and not float and should therefore compact into the pellet after centrifugation. Acid alcohol and ethyl acetate are both much less dense than the other solutions, resulting in the thick pellet that forms after extraction. When looking at the hydrophilic phase, both 10% formalin and acetoacetic buffer are quite dense (1.011 and 1.091) and similar to the relative density of Ascaris eggs. This could account for the eggs being trapped in the disc, as eggs would require time to be pushed down into the pellet. Since the disc forms quickly after the sample is shaken, eggs could have instead adhered to, and been trapped in, the organic matter disc.

Acetoacetic buffer is slightly denser than formalin and would therefore allow eggs to remain in solution, thus being trapped in or above the fatty debris disc. Formalin made up in water is slightly less dense (Manser et al., 2016), thus allowing eggs to settle better when shaken fast or centrifuged and could account for the difference in performance in terms of egg recovery, where formalin samples recovered more eggs than acetoacetic buffer.

The formal–ether concentration was originally developed as a diagnostic method for stool samples to determine total fecal egg counts (Wykoff and Ritchie, 1952). It was then simplified (Ridley and Hawgood, 1956) and optimized (Allen and Ridley, 1970) and is used to concentrate parasites in fresh or preweighed, formalin-preserved fecal samples. Allen and Ridley (1970) stated that the formal–ether concentration, which ideally results in parasites sedimenting at the bottom of the test tube, was preferred over the zinc sulfate (sp. gr. 1.18) flotation method for recovering eggs, where eggs rise to the surface of the supernatant because of a difference in densities between the eggs and the suspension medium. The study also stated that heavier eggs were lost during flotation but were recovered with sedimentation, with the latter resulting in clearer microscopy such that structural details of eggs can be observed more easily and with recovery of a greater number of egg species.

The polarity of a solution determines its solubility. Ethyl acetate is more polar than diethyl ether, causing the latter to be a better emulsifying agent (Manser et al., 2016). Because of the high lipid content of the egg membrane and shell, diethyl ether therefore attracts eggs toward the lipophilic layer and traps them in the disc. Ethyl acetate is a less effective emulsifier, thus resulting in a larger pellet containing more eggs. According to Manser et al. (2016), extractions with diethyl ether resulted in a final deposit that was free of fat, but eggs were trapped in the fats in the debris disc, thus being discarded along with the supernatant layer. Acid alcohol is made up of sulfuric acid, which is polar, and ethanol, which possesses both polar and nonpolar ends, resulting in a more polar solution when combined to form acid alcohol. This could further explain the large deposit formed with the acid alcohol and ethyl acetate combination, as both are less effective emulsifying agents. For fatty sludge samples, ethyl acetate resulted in better egg recovery than diethyl ether (Table I), which could also be due to less effective emulsification of the lipids resulting in reduced trapping of eggs in the disc.

Diethyl ether is flammable, explosive, and hazardous to human health; thus, ethyl acetate is preferred for extraction (Rude et al., 1987). These authors found that a larger plug of debris formed after centrifugation and egg recovery was greater with ethyl acetate as compared with diethyl ether in both instances. Young et al. (1979) reported similar findings, thereby recommending the use of ethyl acetate over diethyl ether. Pakdad et al. (2018) reported a 43.3% Ascaris egg recovery with a 10% formalin and diethyl ether combination. We found that the diethyl ether recovered similar amounts of eggs in primary sludge samples (approximately 45.5%) when combined with both 10% formalin and acetoacetic buffer, but recovery was still far too low. Ethyl acetate performed better when combined with 10% formalin than with acetoacetic buffer, but egg recovery was still too low. Ethyl acetate performed best in combination with acid alcohol (Table I), but the pellet size was not reduced, and microscopy still proved difficult. Replacing diethyl ether with ethyl acetate for phase extraction, even though the latter is less harmful, makes no difference as egg loss is not preventable. Data from this study strongly support the inefficacy of the extraction step that is commonly used in many existing helminth egg enumeration methods.

We support the removal of the extraction step, primarily because of egg loss in the supernatant phase (including the disc of organic matter), and the fact that the combination that produced the best egg recovery (acid alcohol and ethyl acetate: 93.2, 89.8, and 57.3% in water, primary sludge, and fatty sludge, respectively) did not reduce the size of the final pellet for microscopy. This defeats the purpose of the phase extraction step and causes unnecessary exposure of eggs to potentially harmful chemicals without facilitating easier microscopy. We recommend that instead of extraction, samples that have a thick final pellet after the supernatant is removed should be resuspended in a little water and half the sample be analyzed. The egg recovery can then be extrapolated accordingly. This study concludes our work that aimed at optimizing the existing PRG Helminth Method upon comparison with the steps of other helminth test methods. On the basis of data from this study and the previous two, a final optimized method will be renamed the WRDC Helminth Method and published.