Spatial distribution and seasonal profile of parasitic contamination in sewage water samples from the city of Santana do Ipanema, Northeast region of Brazil

Worldwide, it is estimated that 80% of wastewater is released into the environment without proper treatment (UNESCO 2017) and that at least 2 billion people use a source of water contaminated with feces (WHO 2019). Also, there are direct relationships between the prevalence of some parasitic diseases and the presence of their respective etiological agents in water (Yousefi et al. 2006; Omarova et al. 2018).

Among the pathogenic organisms present in wastewater, parasites are the most frequently found (Hatam-Nahavandi et al. 2015). Among the protozoa, stands out: Blastocystis hominis, Giardia spp., Cryptosporidium spp., Entamoeba coli and Entamoeba histolytica. Regarding helminths: Ascaris spp., Trichuris spp. and Hymenolepis nana (Athari 1996; Zahedi et al. 2021). With the exception of E. coli, non-pathogenic protozoa (Khouja et al. 2010), the other parasites usually cause acute gastrointestinal disorders, and can also cause chronic diseases. Consequently, they can also contribute to stunting and other nutritional disorders in children (Thompson & Monis 2004).

The protozoa, Giardia and Cryptosporidium frequently cause waterborne outbreaks (Karanis et al. 2007; Baldursson & Karanis 2011; Efstratiou et al. 2017) and have already been found in the sewers of several countries, including Brazil (Robertson et al. 2000; Montemayor et al. 2005; Santos et al. 2011; Li et al. 2012; Nguyen et al. 2016; Ulloa-Stanojlović et al. 2016). In the country, the presence of E. histolytica, E. coli and geohelminths, Strongyloides spp., Ascaris spp. and Trichuris spp. in wastewaters has also been reported (Paulino et al. 2001; Silva et al. 2014). Due mainly to the presence of strong membranes that protect them from physical and chemical destruction, the protozoa can survive in wastewater for up to 1 month, while helminths can survive for up to 12 months (Stott 2003).

Since the occurrence of parasitosis is determined by spatial and time factors, the characterization of the spatial and seasonal distribution of parasites is an important device to develop strategies to map areas with the potential risk of interaction between humans and parasites, becoming a fundamental instrument for epidemiological surveillance actions (Guimarães et al. 2010; Gomes et al. 2014; Assaré et al. 2015). Something crucial in areas with poor basic sanitation such as the city in question where activities in unsafe waters directly influence the increase of intestinal parasite infections (Ramos 2021). Furthermore, a local coproparasitological investigation on schoolchildren observed a higher proportion of infection by protozoa, 43 cases (89.58%), while 70.8% of positive participants lived in streets with open sewers (Santos et al. 2020a).

As highlighted, the lack of basic sanitization services in that city facilitates the transmission of diseases, especially the dissemination of parasitosis with a direct impact on the quality of life of the local population. Thus, considering the resistance of parasites, which remain viable in wastewater for a long period and the precariousness of the sanitization service in the respective city, the objective of this study was to investigate the occurrence of eggs or larvae of helminth and cysts of protozoa in peridomestic sewage in both the rainy and dry seasons in Santana do Ipanema, Alagoas.

A cross-sectional study was carried out between the months of June, July and October of 2021, using spatial analysis tools, whose units of analysis were the streets of the city Santana do Ipanema.

Santana do Ipanema is a Brazilian municipality belonging to the state of Alagoas. It is the main city of the backcountry of Alagoas, having a population of approximately 47,819 thousand habitants and a territorial extension of 437,875 km² (Brasil 2020a). The Ipanema and Camoxinga rivers stand out among the main water resources that cross the urban center. It has a climate classified as very hot, semi-arid, steppe type, with temperatures above 18 °C in the coldest months. May, June and July are the three consecutive most rainy months, and October, November and December are the three driest (Lopes et al. 2005).

The sample was calculated in the Epi Info™ 6.0 program, for this we used the following parameters: acceptable error of 5%, confidence level of 95%, population of 213 streets and expected prevalence of 3%. The result obtained was 45 streets, with a 10% correction to avoid possible areas without material to collect, which resulted in a final sample of 50 streets.

In the sampling process, a proportional stratification was used to select the streets distributed by the FHP unit, and a simple random sampling was done to compose the sample areas. In this sample composition, the number of streets was proportionally divided taking into account the FHP by which it is served and a proportion was added with correction. A summary of this sampling process is available in the Supplementary Material (see the sampling flowchart used for the selection of the streets of the city of Santana do Ipanema in Figure ES-1).

Two sampling points were established, one at the beginning and the other at the end of the street to collect material in each of the selected areas. All collection sites were georeferenced for map-making.

After establishing the sampling points, the collection was performed over a period of 3 months, the 1st collection being carried out in the rainy season from June to July and the 2nd collection in the driest period, specifically in the month of October. In both, the material was collected only once.

During the collection, it was necessary to homogenize the water by agitation, in order to cause the suspension of the particles. This process was done with the aid of a whisk, and the homogenized material was stored in polypropylene collector bottles with a capacity of 80 ml, packed in a styrofoam box and transported to the Laboratory of Human Parasitology and Malacology of the State University of Alagoas (UNEAL) for processing.

In the laboratory, the sewage samples were processed using the Bailenger Method, modified by Mara & Cairncross (1989), however, with some adaptations. Summarizing, the samples present in the collectors were homogenized. Then the suspension was filtered in conical cups using surgical gauze and the remaining volume of the cup was completed with tap water. This suspension was left to rest for 60 min. At the end of it and with the turbidity that remained, the supernatant was carefully discarded, the water was changed and left to rest for another 60 min. After that, the clear supernatant was discarded and 4 ml of the filtered samples were transferred to 15 ml falcon tubes, which were filled with 6 ml of tap water and centrifuged at 2000 rpm for 3 min. After that, the cloudy supernatant was discarded, leaving only the pellet, which was used to make two slides for each sample, in which Lugol (1%, relative density 1.01) was added to evidence the morphological details of the parasites, and these were analyzed under a binocular optical microscope, using the 100× and 400× magnifications.

The records of the demarcated locations were georeferenced by the Global Positioning System (GPS) with a Garmin eTrex 20 device (Garmin Ltd, Schaffhausen, Switzerland). The collection points were inserted in Qgis software, version 3.18.28 (QGIS Development Team; Open Source Geospatial Foundation Project), where maps of the spatial distribution of positive areas and kernel density estimation were constructed. The analysis using the kernel density estimator allows for highlighting risk areas (hotspots) where there is overlap and agglomeration of cases in a given search radius from a target point (Assaré et al. 2015; Santos et al. 2020b). We considered in our spatial analysis a radius of 100 m and in the construction of the maps a pixel size of 0.0001 was used in the raster output. The geographic grid of the municipality was obtained through the website of the Brazilian Institute of Geography and Statistics (IBGE; https://www.ibge.gov.br).

The data were stored and organized in spreadsheets from Microsoft Excel, version 2019, through which the proportion calculation and table creation were also done.

MBM is characterized by a greater presence of impurities and solids that can obscure eggs and cysts (Ben Ayed et al. 2009). Then we used the surgical gauze for the filtration and retention of these materials that could possibly compromise the analyses, something that proved very useful for the detection of eggs and helminth larvae; however, few protozoa were found. Thus, it is not possible to discern whether this factor is associated only with the low sensitivity of the method, since conditions such as burst, with elimination periods interspersed by non-elimination of cysts by some of these parasitic and low levels in chronic infections of the population (Berne et al. 2014), directly affect the amount of these structures in sewage. Since different researchers revealed the efficiency of MBM in the identification of protozoan cysts in these types of samples (Ben Ayed et al. 2009; Khouja et al. 2010).

The data from this research show that the helminth S. stercoralis was the most frequent in both climatic periods. In addition, there was an increase in the concentration of Hookworms, Metastrongylids and A. lumbricoides, with a consequent reduction in the number of S. stercoralis and Strongylus spp. during the dry season.

Due to the role of soil in the life cycle and transmission, infections by S. stercoralis, Hookworms, A. lumbricoides and T. trichiura are known as soil-transmitted helminthiasis (STH) (Schär et al. 2013). Research involving soil samples found that for STH larvae, the occurrence is higher during rainy months, with eggs being more frequent in the dry months (Oyewole & Simon-Oke 2022), corroborating the findings reported here. This can be explained by the fact that the eggs of some STH are more resistant to environmental conditions, such as hot and dry weather, while the larvae in general suffer from desiccation (Brooker et al. 2006; Amadi & Uttah 2010).

In this way, the high concentration of S. stercoralis larvae in the rainy season can be explained by the lesser exposure to light, which is more intense during the dry season and may have contributed to the reduction of this specimen in this season. The high percentage of A. lumbricoides and Hookworms in the dry period must be related to the local thermal thresholds (20–39 °C), outside of which the surviving of the infectious stages decreases (Seamster 1950; Udonsi & Atata 1987; Alagoas 2018). It is worth mentioning that even in that season, torrential rains can occur, particularly in the month of October and can contribute to increasing humidity (Brasil 2005; Alagoas 2018). Therefore, local climatic factors favor the life cycles of these pathogenic organisms.

STHs in wastewater pose a threat to human health after direct or indirect exposure (Chan 1997; Mara & Horan 2003). The infective larvae penetrate the skin or mucosa (Ancylostoma spp. and S. stercoralis) or when they are inside the egg and are ingested by the host (A. lumbricoides and T. trichiura) (Bethony et al. 2006). Among geohelminths, S. stercoralis stands out for presenting alternation of generations, with a complex life cycle following different routes, including a complete life cycle outside the human host (Jourdan et al. 2017). Although free-living adult worms only live for 2–4 days (Yamada et al. 1991; Conway et al. 1995), this single free-living generation amplifies the number of infective filarial larvae in the environment that can infect humans (Schad 1989; Van Doorn et al. 2007). This versatility in its life cycle may explain the high prevalence of larval forms of this specimen in the analyzed samples.

During the analyses, the parasites Toxocara spp., Strongylus spp., Oesophagostomum spp., Trichostrongylus spp. and Metastrongylids were also detected. The genus Toxocara has important zoonotic parasites. T. canis eggs are highly resistant to physical, chemical and biological factors, surviving for several years in the environment. The accidental ingestion of their eggs present in contaminated water, hands or food can cause serious clinical conditions, such as visceral larva migrans in humans (Despommier 2003; Paquet-Durand et al. 2007; Gakosso et al. 2020).

Strongylus spp. are parasites of equines and donkeys. The high concentration of the parasite during the rainy season is due to the development of larvae until the L3 stage, which can be accelerated if the feces of Equus spp. are moistened by rain, with minimum precipitation of 25 mm being sufficient to increase the density of L3 (Craig et al. 1983; Herd & Willardson 1985; Mfitilodze & Hutchinson 1988). The reduction in the number of parasites in the dry period is due to the effect of desiccation on the amount of infective larvae, which can be more pronounced in areas with a hot and dry climate (Ogbourne & Duncan 1985).

Helminths of the genus Oesophagostomum, occur all over the world and are common parasites of ruminants, swine and some primates. Human infections are rare and may be more common in Africa (O. bifurcum), with some cases reported in Brazil, Malaysia, Indonesia, French, Guyana, China and West Africa (Mehlhorn 2008; CDC 2017).

Species of Trichostrongylus spp. inhabit the digestive tract of ruminants, horses, rabbits and chickens. However, they have been described infecting humans (T. orientalis, T. colubriformis, T. probolurus, T. affinus). Human infections have occurred in some countries (e.g. Iran and Japan) but are rare (Mehlhorn 2008; Taylor et al. 2017).

Metastrongylids are parasites of the respiratory, vascular and nervous systems of mammals and belong to the superfamily Metastrongyloidea (Bowman 2010). Only two species of a family have been confirmed as a cause of disease in humans: Angiostrongylus cantonensis and A. costaricensis, which can cause neurological and abdominal angiostrongyliasis, respectively, with cases reported in the Americas, including Brazil (Morera & Céspedes 1970; Thiengo et al. 2013).

Regarding protozoa, the most frequent were I. butschlii, the complex E. histolytica/E. dispar/E. moshkovskii in the dry season and G. lamblia in the rainy season. Among these, E. histolytica and G. lamblia cause dysentery in humans and animals (Bowman 2010; Taylor et al. 2017). Furthermore, Giardia spp. are transmitted by water, due to their high resistance to chlorine and high infectivity (Putignani & Menichella 2010; WHO 2011; Fregonesi et al. 2012; Monteiro 2017).

In Switzerland, a study of 26 untreated wastewater samples using the application of next-generation sequencing (NGS) to genomic DNA extracted from sewage showed that most of the protozoa detected belonged to the Entamoebidae family (Stensvold et al. 2019), as demonstrated here. However, we did not detect the presence of Endolimax nana, presumably because the survival times of cysts are shorter in natural environments, such as water (Dobell 1943). Regarding the low levels of G. lamblia, several studies showed variations in the concentration of structures of this pathogen in samples of raw effluent and effluent treated in several seasons and stated that the seasonal distribution of the concentration of cysts of G. lamblia in raw sewage varies according to the infection rate of the human population and the climatic characteristics of the study site (Castro-Hermida et al. 2008; Cheng et al. 2009; Nasser et al. 2012).

Spatial analysis allows the identification of patterns of geographic phenomena, where the variable of interest is the very location of events (Medronho et al. 2009). In this sense, the estimation of Kernel density for spatial distribution modeling plays an important role in epidemiology, as it is a simple method to evaluate and estimate the point intensity of an occurrence in a given area and has been widely applied to analyze the epidemiological situation of various morbidities (Gatrell & Bailey 1996; Gomes 2011).

The use of these techniques in the present study allowed the identification of risk areas, which are relative proximity to river areas that may be a consequence of heavy rains that caused flooding by the Ipanema River and Camoxinga River in March 2020, where the city's sewage is also dumped in these water bodies (Brasil 2020c). The flooding phenomenon associated with precarious sanitary sewage facilitates the spread of parasites, especially those associated with zoonotic vectors and reservoirs (Brown & Murray 2013; Huang et al. 2016; Okaka & Odhiambo 2018). Besides that, rain provides essential moisture for the development of eggs into infective larval stages and contributes to their dispersal and larval migration throughout the environment (Oyebamiji et al. 2018). In fact, precipitation can occur in both climatic periods in Santana do Ipanema. Domesticated, stray dogs and synanthropic rodents can also act in the dissemination of parasitic structures, not only in central urban areas, but mainly in peri-urban and semi-rural environments and vice versa (Mustapha et al. 2019; Alegría-Morán et al. 2021).

The contamination of sewage water samples with geohelminths is possibly related to the washing of vegetables before consumption in the city's residences since the water discarded through the sink is generally destined for the sewage network. A survey carried out in the same location with 23 lettuce samples identified that 35% of the analyzed samples were contaminated with the parasites S. stercoralis, G. lamblia and Ancylostoma duodenale (Vieira et al. 2021). Also, an increase in the prevalence of infections by geohelminths associated with direct exposure to wastewater and the consumption of vegetables grown in soil irrigated with it has already been reported in several countries (Amoah et al. 2018).

The predominance of STH may also be associated with the presence of piles of sand used for construction and renovations that are close to the sample collection sites. Where animals (dogs and cats) have a strong impact on the contamination of these places, as they can use them for defecation, and spreading of eggs, larvae, cysts and infectious oocysts contributing to the increase in the number of these parasites in urban waters, since they can be definitive or intermediate hosts (Young & Thackston 1999; Whitlock et al. 2002; Cassenote et al. 2012; Pereira et al. 2016; Kostopoulou et al. 2017).

It is worth noting that we observed the presence of dogs and cats in all areas of our study, and of cattle, horses, pigs, sheep and goats in peripheral areas of the city and in localities near rivers and streams. Fecal contamination by these animals may explain the presence of larvae and eggs of the endoparasites Toxocara spp., Strongylus spp., Oesophagostomum spp., Trichostrongylus spp. and Metastrongylids in sewage.

Dogs and cats are the most important animal hosts for Toxocara spp., especially in developing countries, where most canids and felids have access to public parks and playgrounds, serving as the main source of soil and water contamination with these parasites (Chen et al. 2018). Larval stages of Strongylus spp. are usually found in pastures, their presence in sewage is probably associated with contamination with fecal coliforms by Equus spp. (Ogbourne & Duncan 1985).

The nematodes Oesophagostomum colubianum and Trichostrongylus colubriformis are the ones with the highest prevalence and highest intensity of infection, being considered the nematodes of greatest economic importance for the exploitation of goats and sheep in the Northeast (Costa & Vieira 1984; Silva et al. 1998). Santana do Ipanema economy is based on agriculture, livestock and commerce (Alagoas 2018; Brasil 2020b), partly corroborating with the helminths of veterinary importance discovered in the present study.

The high number of metastrongylids in the drier period, where conditions are often inadequate for the survival of the larvae of some of these specimens (Love 2008), may be linked to water contamination by some mammals such as goats, sheep and pigs that routinely defecate in the open sewer lines, since, due to the scarcity of food during this season, they are released by the farmers on the outskirts of the city and close to water courses. Another concomitant factor is the release of larvae of certain nematodes along the secretions of some gastropods or from the feces of rats that transit through the nets of sewage and act as intermediate and definitive hosts, respectively for some of these pathogens. Additionally in summer, the increase in temperature and torrential rains form the perfect conditions for the reproduction and proliferation of these pests (Mota et al. 2020).

As for protozoa, their detection is inferred to the contamination of sewage water with human fecal material, probably associated with leaking septic tanks, in addition to animal excreta, in which, regardless of the weather period, it is possible to find accumulated wastewater in the streets, which contributes to the concentration and distribution of these organisms.

The use of raw and partially treated wastewater, mainly for agricultural irrigation, is a common practice in some regions of Brazil, being, however, used indiscriminately and without the protection of the health of agricultural workers and consumers (Gobbi 2010). Direct contact with these waters exposes people to S. stercoralis infections and even when wastewater flows to sources of drinking water, such as rivers, it can carry Strongyloides spp. larvae with it (El Shazly et al. 2003). And as already highlighted, the dumping of liquid waste from the sewers is carried out directly in the watercourses of that place, which also receive waste, being used for recreational activities such as bathing and fishing (Brasil 2018), which is another route of infection for people who use these water sources. Furthermore, domestic wastewater flowing into open sewers is two times more likely to cause infection with the E. histolytica/E. dispar/E. moshkovskii complex compared with household waste disposal in closed channels (Junaidi 2021).

In this context, due to poor sanitation, the local population is subject to parasitic infections, especially helminthiasis and helminthic zoonoses, as sewage water exposed to the environment contributes to the maintenance and propagation of parasites between animals and humans in the city in question. Where spatial analysis and kernel estimator have been shown to be vitally important tools to identify priority areas, information that can be used to promote strategies for intervention actions by local agencies aimed at controlling these parasites and improvements in sanitation, such as the proper disposal of liquid waste. It is also emphasized that it is essential to need further studies aimed at the use of other techniques to verify the effectiveness of MBM with the adaptations described here in order to identify these organisms, especially protozoa.

The authors state that there were no proposals for financing public, private and philanthropic agencies.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.