Inactivation of Ascaris for thermal treatment and drying applications in faecal sludge

Ascaris Lumbricoides is the most common helminth of human health importance, and the most resilient helminth found in faecal sludge. There are numerous types of sludge treatments; however, heating and drying are most commonly used for pathogen inactivation. Ascaris suum eggs were heated in a water bath at 40 – 55 (cid:1) C for 10 seconds to 60 minutes in water, as well as heated in both urine diversion dry toilet and ventilated improved pit latrine sludge at 40 (cid:1) C, 60 (cid:1) C and 80 (cid:1) C for times ranging from 5 seconds to 120 minutes. Eggs were also spiked into sludges of different moisture contents and incubated over 12 weeks at 25 (cid:1) C, with samples analysed weekly. Overall, we concluded that eggs were inactivated at temperatures > 50 (cid:1) C, that the temperature – time relationship directly impacted ef ﬁ cacy of heat treatment, that suspension medium had no effect, and that eggs survived better in wet rather than dry sludges.


INTRODUCTION
Approximately 2.3 billion people globally lack access to basic sanitation facilities and one-third of the world's population is infected with soil-transmitted helminths (STHs) (JMP ; Cooper & Hollingsworth ). Ascaris lumbricoides, also known as the human roundworm, is the most common STH of human health importance (Brownell & Nelson ). Infection with A. lumbricoides is most prominent in areas that lack a potable source of water, improved sanitation and proper hygiene practices, with an estimated 804 million people infected worldwide (Jourdan et al. ).
Mild infections may be asymptomatic; however, heavy worm infections can lead to symptoms such as diarrhoea, bloating, abdominal blockages and discomfort, malnutrition, and impaired growth and cognitive development (Cooper & Hollingsworth ). Diarrhoeal diseases are the cause of 1.3 million deaths per year, of which one in eight are children under the age of five years (Kotloff ).
Ascaris eggs can withstand harsh environmental conditions and are considered the most resilient organisms found in faecal sludge, as they are able to survive for up to seven years in the soil (Pecson & Nelson ). Ascaris spp. eggs are therefore commonly used as indicator organisms of faecal contamination and for inactivation experiments (Maya et al. ).
Heat treatment technologies for sludge have become common practice due to increased pathogen inactivation success (Belcher et al. ). There have been a number of studies globally that have focused on heating or drying for pathogen inactivation and sludge sanitisation (Brownell & et al. ; Andes & Paller ). Composting systems and drying beds are feasible treatment options that operate at lower temperatures over prolonged periods of time, and are common practice in Africa. Koné et al. () reported that heat generation due to the composting process in a drying bed resulted in 90-100% Ascaris inactivation after 80 days at !55 C. Septien et al. ()  () that focused on heat treatment at 60-80 C in water.
Eggs were heated in water at 40, 45, 50 and 55 C for times ranging from 10 seconds to 60 minutes.
According to Jebri et al. (), the suspension medium of the eggs plays a role in the efficacy of heat treatment. Buttar et al. () went on to report that sludge, or any similar suspension medium, may act as insulation against heat, thus higher temperatures and longer exposure times might be needed for successful inactivation. The role of the suspension medium on the inactivation of Ascaris eggs was therefore investigated in this study, testing water, urine diversion dry toilet (UDDT, to be referred to as UD from henceforth) sludge and ventilated improved pit (VIP) latrine sludge where eggs were heated at 40, 60 and 80 C, for various exposure times that were selected based on previous data (Naidoo et al. ).

Sample collection and sludge characterisation
The effects of heating were tested in three separate experiments. UD sludge was collected from a stockpile that was collected from UD toilets within the eThekwini Municipality (Durban, South Africa) and transported to the Isipingo Wastewater Treatment Plant in KwaZulu-Natal for treatment. VIP sludge was collected directly from household latrines in Bester (within the eThekwini Municipality), and wet VIP sludge was collected directly from the vacuum trucks that were emptying pits in the Bester area at the time of sampling. Water was added to the UD sludge to match the moisture content of the wet VIP sludge, for the purposes of consistency during treatment. The sludge characteristics are summarised in Table 1. Ascaris suum eggs were purchased from Excelsior Sentinel Inc. (USA) and were stored at 4 C until needed.

Experiment 1: heat treatment of eggs in water
The first experiment was a continuation of Naidoo et al.
Eggs were heated at 40, 45, 50 and 55 C for 10 and 30 seconds, and at 1, 2, 5, 10 and 60 minutes. Plastic 15 mL test tubes containing tap water were preheated in a water bath to the respective test temperature. Approximately 500 Ascaris eggs (suspended in 1 mL of water) were spiked into each heated test tube and exposed for the test time. The tubes were then removed from the water bath and the contents brought back to room temperature immediately by emptying into iced tap water, in order to prevent prolonged heat exposure. Samples were treated, processed and analysed using light microscopy, both before and after incubation according to the methodology used by Naidoo et al. ().

Experiment 2: The effects of the suspension medium on the efficacy of heat treatment
The second experiment involved the heating of Ascaris eggs in both VIP and UD sludge, and water as a control. Test parameters included 30, 60 and 120 minutes at 40 C, 30 seconds, 2 minutes and 5 minutes at 60 C, and 5 and 10 seconds and 1 minute at 80 C. The UD sludge was diluted to match the moisture content of the wet VIP sludge.
Sludge samples of approximately 10 g each were weighed out into aluminium cups (70 mm × 40 mm) and covered with sheets of aluminium foil. The water bath was preheated to the respective test temperature, after which the aluminium cups containing the sludge were inserted into holes cut into a polystyrene sheet (350 mm × 250 mm × 15 mm), being fully immersed in the water. Once the entire system reached the test temperature, eggs were spiked into the sludge and mixed, and the cups were immediately covered again to prevent moisture loss. The samples were exposed for the respective test times, after which the cups were removed and placed into plastic bowls containing iced water, and approximately 30 mL iced water was poured into the sludge sample and mixed to allow the sample to return to room temperature and prevent prolonged heating.
The samples were then processed according to the PRG Helminth Method (PRG website). Ammonium bicarbonate was poured into the aluminium cups until the sludge was just covered and mixed well. The contents of the cup were then poured over a set of drum sieves (100 μm over a 20 μm sieve). The sample was washed thoroughly in the sieve, using pressure from a hose on the tap, and by breaking any clumps using the back of a gloved hand. The 100 μm sieve was then removed and the retentate discarded. The retentate (containing the Ascaris eggs) on the 20 μm sieve was then washed thoroughly and collected into four plastic 15 mL test tubes. These were centrifuged at 1,512 × g (3,000 rpm) for 10 minutes and the supernatant discarded.
Zinc sulphate (ZnSO 4 ) was added to each tube in 3 mL aliquots to a total of 14 mL while vortexing to break up the pellet and homogenise the suspension. The test tubes were then centrifuged at 672 × g (2,000 rpm) for 10 minutes, to allow eggs to float up into the liquid column above the sediment. The supernatant was then poured onto a smaller 20 μm sieve, washed with water, and collected into a single 15 mL tube. The final samples were centrifuged at 1,512 × g (3,000 rpm) for 3 minutes, after which the supernatant was discarded and the final pellet microscopically analysed. The samples were then washed back into the test tubes, incubated for 28 days at 25 C and re-analysed microscopically. Eggs were scored and categorised according to morphology and viability, as per Naidoo et al. ().    (Figure 1(a) and 1(b)). Treatment at 50 and 55 C led to a statistically significant egg inactivation after 60 minutes (p < 0.001) and 5 minutes (p < 0.001) of exposure, respectively. At 50 C for 10 minutes of heat exposure, egg viability before incubation was 85.5%, and a decline in viability was seen after incubation (65.5%;

RESULTS AND DISCUSSION
p ¼ 0.762), but this did not meet the inactivation criterion for this study. After 60 minutes of heat exposure, egg viability was 86.5% before incubation and 1.9% after incubation (p < 0.001). Treatment at 55 C rendered an egg viability of 89, 92 and 84% before incubation, and 3.8, 0.2 and 0% after incubation, for 5 minutes (p < 0.001), 10 minutes (p < 0.001) and 60 minutes (p < 0.001) of heat exposure, respectively, meeting the inactivation criterion for this study (Figure 1(d)).
The 5 C difference between heat treatment at 50 and 55 C appears to be crucial in terms of inactivation efficacy, as the most effective exposure time for each temperature ranges from 60 down to 5 minutes. It should also be noted that complete inactivation was not observed after 1 hour of treatment at temperatures below 50 C.  where an egg sometimes appears somewhat damaged, but upon incubation, it would develop further, indicating that inactivation was not successful.

Experiment 2
Results from the nested ANOVA (Table 2) for Experiment 2 indicated that alone, temperature had a significant effect on sludge (65.5%; p ¼ 1.000), thus none of the above met the inactivation criterion for this study (Figure 2(a)). Figure 2(b) shows that significant inactivation was achieved at 60 C after 30 seconds of exposure in water (5.6%; p < 0.001), UD sludge (12.7%; p < 0.001) and VIP sludge (3.6%; p < 0.001). A near complete inactivation of Ascaris eggs was obtained at 2 minutes' exposure (p < 0.001) and complete inactivation was achieved at 5 minutes' exposure (p < 0.001), at 60 C in all three suspension media. At 80 C, significant inactivation was achieved after 5 seconds' exposure in water (3.3%; p < 0.001), UD sludge (0.0%; p < 0.001) and VIP sludge (3.6%, p < 0.001). After 10 seconds of treatment, egg viability was almost negligible and at 60 seconds, complete die-off was observed (Figure 2

Experiment 3
Results from the nested ANOVA (Table 2)

CONCLUSION AND RECOMMENDATIONS FOR FUTURE WORK
The conclusion and recommendations for future work are as follows: • This study was aimed at determining the effects of low temperatures, suspension medium and storage conditions on Ascaris eggs.
• Heat treatment at 40 and 45 C after 60 minutes, and treatment at 40 C after 120 minutes were insufficient for Ascaris inactivation, providing critical data for the planning related to slow heating technologies such as drying beds.
• At 50 and 55 C, 60 minutes and 5 minutes, respectively, are sufficient for successful Ascaris inactivation.
• Furthermore, morphological damage was not necessarily visible before incubation. The incubation step is therefore imperative when testing any kind of heat treatment in order to verify inactivation.
• The suspension medium did not play a role in the efficacy of heat treatment of the eggsinactivation patterns were the same for water and UD and VIP sludge, meaning that heat treatment of Ascaris eggs can be done in water rather than having to source sludge • Time had an effect on Ascaris egg survival and development in sludge, indicating the potential for egg inactivation during long-term storage.
• In combination with moisture content, a significant effect was also observed. Eggs preferred wetter sludges for development, with 50% moisture being the optimum condition, and development was much slower in drier sludges (20-30% moisture).
• Fungal growth is inevitable over time, supporting egg inactivation during long-term storage of sludge.