Soil-transmitted helminth eggs assessment in wastewater in an urban area in India

Water quality and sanitation are inextricably linked to prevalence and control of soil-transmitted helminth infections, a public health concern in resource-limited settings. India bears a large burden of disease associated with poor sanitation. Transformative onsite sanitation technologies are being developed that feature elimination of pathogens including helminth eggs in wastewater treatment. We are conducting third-party testing of multiple sanitation technology systems in Coimbatore (Tamil Nadu) India. To ensure stringent testing of the pathogen removal ability of sanitation technologies, the presence of helminth eggs in wastewater across the town of Coimbatore was assessed. Wastewater samples from existing test sites as well as desludging trucks servicing residential and non-residential septic tanks, were collected. The AmBic methodology (based on washing, sieving, sedimenting and floating) was used for helminth egg isolation. We tested 29 different source samples and found a 52% prevalence of potentially infective helminth eggs. Identification and enumeration of helminth species is reported against the septage source (private residential vs. shared toilet facility) and total solids content. Trichuris egg counts were higher than those of hookworm and Ascaris from desludging trucks, whereas hookworm egg counts were higher in fresh wastewater samples. Surprisingly, no correlation between soil transmitted helminth eggs and total solids was observed.


INTRODUCTION
Soil transmitted helminth (STH) infections are a public health concern in resource-limited settings with poor sanitation. While mortality is low, morbidity due to helminth infections is more insidious. Malnutrition, abdominal cramps, diarrhea, and long-term effects like reduced cognitive and intellectual ability, are common problems, particularly in children <5 years for whom consequences are life-long (Bartram & Cairncross ).
Helminths reproduce via eggs and sometimes larvae, which are the infective agents transmitted via the fecaloral pathway (e.g. Ascaris lumbricoides and Trichuris trichiura) or the skin (e.g. hookworm species and Strongyloides stercoralis). Poor sanitation and drinking water quality are significantly associated with risk of infection (Echazú et al. ). An effective treatment system must protect people from contact with their excreta and explicitly break the cycle of infection with STHs by ensuring that effectively eliminate or inactivate pathogens, including helminth eggs with a target <0.1 egg/L (IWA ).
India bears a large burden of disease, harboring nearly 25% of global cases, with 220.6 million children in need of preventive chemotherapy (Salam & Azam ). We are conducting third-party testing of multiple Reinvent-the-Toilet systems in the city of Coimbatore, in the southern Indian state of Tamil Nadu. In order to assess the effectiveness of sanitation technologies in removing or inactivating helminth eggs from treated waste, it is necessary to gather information on prevalence, species and viability of STHs in this area where sanitation system testing sites may be located.
Ascaris lumbricoides produces the most resistant eggs, and is also one of the commonest STHs in many developing countries, including Peru, where it was recently found to be most prevalent in domestic wastewater (Yaya-Beas et al.

).
A study on the prevalence and diversity of helminth eggs in pit latrine sludge in Yaounde, Cameroon, found an average of 80 eggs/g dry matter, with Ascaris and four other species, including hookworm, being prevalent (Nzouebet et al. ). In a recent review of 39 studies that reported the prevalence of STH infections from 19 different states in India, Ascaris lumbricoides was the most prevalent parasite with >50% prevalence reported from six states, including Tamil Nadu (Salam & Azam ). Almost 90% of the studies in Salam & Azam () reported the prevalence of more than one parasite species in the same sample population.
We conducted an exploratory assessment on the presence of helminth eggs in wastewater in the town of Coimbatore by collecting samples from sanitation system test sites as well as randomly sampling from desludging trucks servicing residential and non-residential septic tanks. It was our intent to determine the presence of helminth eggs in these wastewater sources, identify the species found, and determine egg viability or lack thereof. We adopted the methodology for extracting STH eggs from wastewater and sewage sludge developed by Hawksworth and Archer (Trönnberg et al. ) and modified by Archer (Pebsworth et al. ), known as the AmBic method. This method follows four main principles: (1) washing, (2) sieving, (3) flotation, and (4) sedimentation. The USEPA method (Yanko ) is based on the same principles but is much longer and includes an additional extraction step using acetoacetic buffer and ethyl acetate. As ethyl acetate is a solvent which may affect the protective properties of helminth egg walls, this step is only used following the AmBic method when it is absolutely necessary and when the assessment of viability is not required. Depending on the nature of samples obtained, the AmBic method may be adapted to accommodate them. Thus, very clear water samples may only need to be filtered and centrifuged to sediment a small deposit for microscopic observation, whereas the extraction step may need to be added at the end of the AmBic method if the final deposit is very thick and would otherwise require a very large number of slides to be prepared for microscopy.
This study reports on the presence of helminth eggs in wastewater, assessed in 29 samples from different sources in Coimbatore. We identified and enumerated helminth species eggs, both dead and potentially viable (PV), and examined these results in relation to wastewater source categories (e.g. single family homes versus shared toilets) and a physicochemical wastewater parameter of total solids content.

Description of source and sampling
A sample size of convenience was used in this exploratory study. Twenty-six fecal sludge samples from randomly selected desludging trucks and three fresh wastewater samples from an apartment complex and a shared toilet were analyzed (n ¼ 29 total samples). A stopwatch was used to measure the amount of time that it took the truck to discharge, and grab samples were taken every 30 s (starting with initial discharge) as the truck was emptying using 5 L containers. Three 5 L grab samples, representing the beginning, middle, and end of emptying, were combined to make a 15 L composite sample for each truck. A 2 L sub-sample of the composite sample was reserved for helminth egg screening, 4.5 L was reserved for determining the percentage solids content and the remainder was stored. Information related to the source of the sample (location address, property type, type of sanitation system, inclusion of greywater) was also collected from truck drivers.

Fresh wastewater samples
Property type included residential (either houses or apartments) sites, commercial sites (e.g. a factory) and public sites (e.g. school, police station). Inclusion of greywater was tracked, since in Coimbatore typically only blackwater from toilets goes into septic tanks, while greywater is drained separately to a soak field. The type of sanitation system describes either septic tanks or storm drains and soak pits.

Helminth egg isolation and enumeration
For helminth eggs assessment, 2 L volumes were obtained for all samples. Some samples had visibly higher solids content than others, consequently sub-samples of 250, 500 or 1,000 mL were tested rather than the full 2 L sample. The 26 samples collected from desludging trucks were partially processed and stored for analysis to be conducted at a later time; the fresh samples collected from the shared toilet and apartment complex were analyzed immediately.
The preparation was as follows (see Figure   In order to continue processing the stored samples, they had to be washed twice in distilled water to remove the formalin and all supernatant fluid was discarded. The deposit remaining in a 15 ml tube was required to be 1 mL and in a 50 mL tube 5 mL. So, when this was not the case, the deposit was shared among two or more tubes to accommodate this. The flotation step was then performed using zinc sulfate (ZnSO 4 ) at a specific gravity (SG) of 1.3 to allow for all STH eggs to float: each tube was filled to the 14 mL/45 mL mark while vortexing them with an applicator stick in the tube to act as a stirring rod. Next they were centrifuged at 617 g to float the eggs. The entire supernatant fluid was poured over a 20 μm pore, 100 mm diameter stainless steel pan sieve and washed well with distilled water from the fertilizer pump.
All the filtrand was then returned to one or more test tubes and centrifuged at 1,389 g to obtain a deposit and the supernatant fluid was discarded. If the deposit was in a quantity small enough to easily transfer to one or two microscope slides, then this was the final step of sample preparation.
If the deposits were a larger quantity, then the extraction step was added. This step involved the addition of 7 mL acetoacetic buffer (15 g sodium acetate tri-hydrate plus 3.6 mL glacial acetic acid made up to 1 L with dis- % Solids ¼ Dry mass (g) À mass of petri dish (g) Initial sample volume (mL) Ã100% (3)

Identification of STH eggs
Human helminth eggs found in the desludging truck samples were those of the three commonest STH species, namely Ascaris, Trichuris and hookworm, as well as two tapeworms, Hymenolepis nana and H. diminuta. Other eggs present were Aspiculuris sp., Heterakis spumosa and Trichosomoides crassicauda (common rat parasites) and Calodium hepaticum, a liver parasite of mammals very common among rats (old name, Capillaria hepatica). Egg counts of PV eggs of individual species ranged from <1 to 56 eggs/L.
At the shared toilet, two fresh sewage samples were col-sample was taken from the apartment sump. A 500 mL sub-sample was processed and examined and 2 dead Ascaris eggs plus 52 hookworm sp. eggs/L were found.
All egg counts were converted to eggs per liter (Table 1 when only PV pathogenic spp. eggs were considered, Trichuris and hookworm (28% each) were highest, followed by Ascaris (14%). There was a similar trend when all eggs were included (Figure 3(a)).
For illustrative purposes, samples with egg counts were equally distributed into three categories by egg concentration (0-1, 1-5, and >5 eggs/L) (Figure 3(b)).  (Figure 4(b)), a factor of 10 and 100 times higher than for the desludging truck samples. Figure 4(b) is therefore plotted on a log scale and it also includes the maximum recorded concentration value for each species.

Effect of the septage source
We further examined the egg prevalence in desludging trucks only. A prevalence of 77% (n ¼ 26) across these samples was found when both dead and PV pathogenic eggs were considered (the prevalence was 50% when only PV pathogenic eggs were counted). The average The majority of the 26 samples (n ¼ 14) came from houses, followed by shared toilet facilities (n ¼ 5). Positive samples were found across all sources, although many samples from houses and shared toilet facilities tested negative for helminth spp. eggs (Figure 5(a)). The average concentration of PV helminth spp. eggs was highest in the shared toilet and drainage samples ( Figure 5(b)); however, average concentration counts were not high (<15 egg/L) and the maximum recorded concentration (44 egg/L) was from a house.

Effect of septage solids content
Total solids content was evaluated for the samples collected from the desludging trucks and was found to be highly variable, ranging from <0.1% to 7%.  For graphical illustration of results, the samples were divided into three categories according to their solids content to give approximately equal numbers of samples across each: 'low' if the solids content was <1% (n ¼ 10), 'medium' if the solids content was between 1 and 3% (n ¼ 7), and 'high' if the solids content was >3% (n ¼ 9).
Helminth egg prevalence among each category of total solids content was similar to the overall prevalence for the desludging truck samples (Figure 6(a)), indicating that prevalence was unrelated to solids content.
The average concentration was low (<10 eggs/L) across all categories, and also did not depend on solids content ( Figure 6(b)). The highest concentration recorded was 44 eggs/L. This sample had a solids content of 1.2% and was from a single-family house.

DISCUSSION
This study explored the prevalence and concentration of STHs in wastewater samples in an Indian urban area, the city of Coimbatore, one of the sites where Reinvent the Toilet Technologies are to be tested. For assessing the ability of sanitation technologies to achieve biologically safe treated waste, it is important to additionally assess the viability of the eggs. We determined viability by using rigorous criteria including morphological analysis and presence of visible larvae as observed by an experienced microscopist.
We recorded a 52% prevalence of samples with PV and therefore infective helminth eggs in 29 samples from different sources. A higher prevalence of 72% was obtained if we included both dead and PV eggs. Nine different species of helminth eggs were identified in this study. We note that the isolation process adopted a solution with specific gravity SG ¼ 1.3 for the flotation step, a SG value well suited to recover all species of helminth eggs with relative densities ranging between 1.06 and 1.24 g/cm 3 (da Rocha et al. ). In East Africa, Ascaris, Trichuris and hookworm sp. were reported as equally prevalent (Brooker et al. ). We, however, found that Trichuris was the commonest helminth as defined by the number of samples with > 5eggs/L ( Figure   3(c)), and highest average count by species (Figure 4(a)).
Hookworm sp. eggs were present in the highest concentration (up to 49,000 eggs/L) in fresh wastewater samples from the shared toilet (Figure 4(b)). Although Ascaris eggs were also present, they were not the main species. It is possible that hookworm is the most prevalent species in infected individuals but not in samples obtained from septic tanks.
We hypothesize that hookworm sp. eggs are (1)  We analyzed the effect of the wastewater source on the prevalence of helminth eggs to evaluate whether shared facilities may be more likely to be positive for STHs than individual houses; however no effect was recorded and positive samples and counts were similar.
Due to their larger size, helminth species eggs are known to adhere to solid surfaces and settle more easily in sludge than bacteria and viruses (Shamma & Al-Adawi ). STH eggs are therefore expected to be in higher concentrations in samples with higher total solids derived from settled sludge. We measured the total solids in desludging truck samples and found a large variability in solids content, consistent with observation by others in desludging trucks from a different city in Tamil Nadu (Krithika et al. ).
This variability is attributed to many factors including the suction power of the vacuum pump on the truck, the collection of supernatant from the septic tank rather than the sludge, and different levels of sludge accumulation at the source (as determined by different cleaning frequencies).
The prevalence and concentration values of STH eggs do not vary significantly across different solids contents ( Figure 6(a) and 6(b)).
The lack of correlation between helminth egg presence and total solids is surprising. It has been observed that solids interfere with STH recovery (Amoah et al. ); however we believe this was not the case in this study for two reasons: (1) the total solids values below 5%the range in our studyare generally not considered problematic (Amoah et al. ); and (2) the AmBic method is wellsuited to isolate eggs from soil particles and is therefore effective for samples with high solids content. We therefore believe that the reported data reflect the actual content of the samples and that the health risk posed by fecal sludge from septic tanks in this area is independent of the solids content of the sample and is therefore also independent of whether the trucks collect from the sedimented sludge or the supernatant of the septic tank.
In conclusion we report that STH eggs are common in wastewater in the urban environment selected as a test site for Reinvent-the-Toilet technologies in southern India. The described approach to isolation, enumeration and viability assessment of STH eggs is well suited to determine the effectiveness of waste treatment by transformative sanitation technologies that will be a key tool towards eradication of these infections.