Fate of Cryptosporidium and Giardia through conventional and compact drinking water treatment plants

Over the past three decades, a notable rise in the occurrence of enteric protozoan pathogens, especially Giardia and Cryptosporidium spp., in drinking water sources has been observed. This rise could be attributed not only to an actual increase in water contamination but also to improvements in detection methods. These waterborne pathogens have played a pivotal role in disease outbreaks and the overall escalation of disease rates in both developed and developing nations worldwide. Consequently, the control of waterborne diseases has become a vital component of public health policies and a primary objective of drinking water treatment plants (DWTPs). Limited studies applied real-time PCR (qPCR) and/or immunofluorescence assay (IFA) for monitoring Giardia and Cryptosporidium spp., particularly in developing countries like Egypt. Water samples from two conventional drinking water treatment plants and two compact units (CUs) were analyzed using both IFA and qPCR methods to detect Giardia and Cryptosporidium. Using qPCR and IFA, the conventional DWTPs showed complete removal of Giardia and Cryptosporidium, whereas Mansheyat Alqanater and Niklah CUs achieved only partial removal. Specifically, Cryptosporidium gene copies removal rates were 33.33% and 60% for Mansheyat Alqanater and Niklah CUs, respectively. Niklah CU also removed 50% of Giardia gene copies, but no Giardia gene copies were removed by Mansheyat Alqanater CU. Using IFA, both Mansheyat Alqanater and Niklah CUs showed a similar removal rate of 50% for Giardia cysts. Additionally, Niklah CU achieved a 50% removal of Cryptosporidium oocysts, whereas Mansheyat Alqanater CU did not show any removal of Cryptosporidium oocysts. Conventional DWTPs were more effective than CUs in removing enteric protozoa. The contamination of drinking water by enteric pathogenic protozoa remains a significant issue globally, leading to increased disease rates. Infectious disease surveillance in drinking water is an important epidemiological tool to monitor the health of a population. Supplementary Information The online version contains supplementary material available at 10.1007/s00436-023-07947-8.


Introduction
Water-related diseases are responsible for the highest number of deaths and diseases worldwide, according to the World Health Organization (WHO), with more than 3.4 million fatalities each year. Children account for around 1.4 million of these deaths (Abuseir 2023). In terms of causing fatalities, the lack of clean and safe drinking water along with inadequate sanitation surpasses war, terrorism, and weapons of mass destruction combined (Abuseir 2023). Water-related illnesses, such as gastrointestinal infections, diarrhea, and systemic diseases, lead to an annual economic loss of approximately US$ 12 billion globally (Alhamlan et al. 2015). Protozoan parasites like Cryptosporidium spp., and Giardia spp., are from the most frequently identified as the cause of diarrheal outbreaks in both Section Editor: Yaoyu Feng 1 3 developed and developing countries (Karanis et al. 2007;Gad et al. 2019;Al-Rifai et al. 2020). The right to access safe water, which is essential for the survival of living beings, has been acknowledged as a universal human right (Taviani et al. 2022). Consumption or exposure to contaminated water can lead to the transmission of various pathogens, including enteric bacteria, viruses, and parasites, causing severe diseases that pose a significant global public health concern (WHO 2019). So, it is crucial to monitor drinking water for pathogens to protect human and animal health.
Waterborne outbreaks caused by protozoa contamination are a significant concern. Cryptosporidium and Giardia species are particularly notable as they can survive in aquatic environments despite the use of chlorine disinfectants (Elmehy et al. 2021). Regrettably, many people worldwide do not have access to safe water, which is free from harmful pathogens and contaminants. This issue is a significant public health concern, even for developed countries (McKee and Cruz 2021). Protozoan parasitic diseases transmitted through contaminated water are found globally and have caused both epidemic and endemic infections in developing countries (Cotruvo et al. 2004;Baldursson and Karanis 2011). For the past four decades, there has been a significant global focus on Cryptosporidium and Giardia species due to their ability to cause waterborne and foodborne illnesses (Mahmoudi et al. 2017;Rosado-García et al. 2017). These protozoa are excreted through feces and have a remarkable resistance to environmental factors, such as high temperatures, chemical water disinfectants, and dehydration (King and Monis 2007).
In 2012, the prevalence of giardiasis in Europe was 5.4 cases per 100,000 population. As for cryptosporidiosis, the prevalence was 10.5 for females and 13.8 for males (ECDC 2014). Unfortunately, there is a lack of comprehensive data on the prevalence of cryptosporidiosis and giardiasis in Egypt and only a limited number of environmental studies focusing on Cryptosporidium and Giardia have been identified (El-Kowrany et al. 2016;Gad et al. 2019;Rizk et al. 2019;Hamdy et al. 2019). The main objectives of this study were (1) to assess the efficacy of different drinking water treatment methods in removing Cryptosporidium and Giardia parasites, which pose risks to human health, (2) to quantify the concentration of these parasites using qPCR and IFA detection methods, both of which were effective in identifying Cryptosporidium and Giardia in water samples, and (3) to investigate the prevalence of these parasites in both raw and treated water samples.

Sampling and DWTPs descriptions
For the present study, four DWTPs were selected. Among them, two were conventional DWTPs catering to large city communities, while the other two were small CUs serving comparatively smaller communities (Fig. 1). The two conventional DWTPs are Shubra Alkheymah, which produces 46300 m 3 of drinking water per day and serves 2.5 million persons, and is located in the Shubra Alkheymah district. Shubra Alkheymah DWTP is composed of an intake system that is supplied with raw surface water from the mainstream of the Nile River, a distribution well, 12 clarifiers (working with a pulsator), 34 rapid sand filters, and a drinking water storage tank. The second conventional plant is Imbaba DWTP, which produces 1.43 million m 3 of drinking water per day and serves people in Al-Remayah, Al-Baragil, Al-Khalayfa, Nahia, Ezbet Al-Eseely, Imbaba, Al-Waraq, and Al-Kitkat districts. The intake of Imbaba DWTP is supplied with raw surface water from the mainstream of the Nile River. Imbaba DWTP is composed of an intake system, distribution well, 20 clarifiers (working with a pulsator), 80 rapid sand filters, and a drinking water storage tank (Fig.S1).
The study also included two drinking water treatment compact units (CUs): Niklah CU and Mansheyat Alqanater CU. Niklah CU has a daily drinking water production capacity of 2040 m 3 , and its freshwater supply is sourced from El-Nasery canal, which branches off from the Nile River. Mansheyat Alqanater CU, on the other hand, produces 2000 m 3 of drinking water per day and is continuously supplied with fresh water from El-Behery canal, which also branches off from the Nile River. Both Niklah and Mansheyat Alqanater CUs share a similar design, consisting of an intake system and a compact unit where clarification and sand filtration processes take place. The treatment process concludes with the final product being stored in a drinking water tank (Fig. S2).
A total of 96 samples were collected from the inlet (raw freshwater) and outlet (final treated drinking water) of two conventional DWTP (i.e., Shubra Alkheymah and Imbaba) and two CUs (i.e., Niklah and Mansheyat Alqanater) within Greater Cairo, Egypt (Fig. 1). Each sample was collected in duplicate, one (10 L volume) of them was used for IFA technique and the other one (10 L volume) for qPCR technique. The samples were collected monthly at the same time from each sampling site for 1 year from February 2019 to January 2020.

Immunofluorescence assay
Each sample was filtered through sterile nitrocellulose membranes (142 mm diameter and 0.8μm pore size) using a sterilized stainless steel pressure filtration system (Millipore). The nitrocellulose membrane filter was removed from the filter housing and transferred into a suitable clean glass Petri dish (142mm diameter). About 25mL of eluent (0.1% Tween 80) was gently poured on the surface of the membrane filter to facilitate the detachment of particulate material from the membrane (ISO/FDIS 15553:2006). The last step was repeated and the obtained washing solution was subjected to centrifugation at 1500 ×g for 15min. The supernatant was aspirated and the obtained pellet was resuspended in 10 mL phosphate buffer saline (pH = 7.4), vortexed from 10 to 15 s, and transferred into a Leighton tube. One milliliter of the 10X SL-buffer-A and 1 mL of the 10X SLbuffer-B were added to a Leighton tube. Then, 100 μL of the resuspended Dynabeads Cryptosporidium and 100 μL of the resuspended Dynabeads Giardia (Dynabeads™ GC-Combo, ThermoFisher Scientific, USA) were added to the solution in Leighton tube. The immunomagnetic separation and immunostaining (DAPI and FITC) steps were conducted according to EPA Method 1623(2005 and ISO/FDIS 15553:2006. The positive controls for both parasites (AccuSpike™-IR, Waterborne™, Inc., USA) were used along with each batch of samples for confirming the productivity of the method.

Quantitative real-time PCR assay
Similar concentration steps to IFA until obtaining the solution from the dissociation of the Dynabeads-cysts/oocysts complex were applied for qPCR. The immunomagnetic purified samples were subjected to extraction of environmental DNA using the DNeasy PowerLyzer PowerSoil Kit (QIAGEN, USA) according to the manufacturer's instructions. The uniplex qPCR assay was performed to quantify the target protozoa in the samples; a qPCR reaction was performed in a 20-μL reaction volume using a QuantiNova syber green qPCR kit (Qiagen, Germany). The reaction mixture was composed of 5 μL of the DNA template, 10 μL of the master mix, 0.5 μL from each primer (forward and reverse) for Cryptosporidium (Haque et al. 2007) and Giardia intestinalis (Guy et al. 2003), and 4 μL of Nucleasefree water. The PCR temperature conditions were 95°C for 10 min and 45 cycles of 15 s at 95°C and 1 min at 60°C. Nuclease-free water was also included in each run as a negative control. Absolute quantification of gene copy (GC) was performed by comparing cycle threshold (Ct) values to the DNA standard, which was included in every qPCR run (BIO-RAD, CFX96 Rial-Time System, USA). The DNA standards for Giardia and Cryptosporidium were prepared from AccuSpike-IR (Waterborne™, Inc., USA). The DNAs were extracted using the DNeasy PowerLyzer PowerSoil Kit (QIAGEN, USA). The PCR reactions were performed separately for both Giardia and Cryptosporidium. The obtained PCR products were purified using GeneJET PCR Purification Kit (Thermo Scientific, Lithuania). Nucleic acid concentrations of the purified PCR products were determined by NanoDrop Fluorospectrometer (Thermo-Scientific, USA). The number of DNA copies was determined by multiplying the DNA concentration by Avogadro's constant and dividing by the product size and average weight of a base pair as in the following website https:// cels. uri. edu/ gsc/ cndna. html (Rizk and Hamza (2021). The standard curves of each examined organism were separately prepared by tenfold serial dilution of the nucleic acid standard ranging from 5×10 1 to 5×10 6 copies/reaction. The limits of detection for the assay were determined as more than or equal to 10 GC/reaction.

18S rRNA high-throughput amplicon sequencing analysis
The DNAs of eight samples were selected randomly to cover the inlet and outlet of different DWTPs and CUs during the course of the study. The hypervariable V4 region of eukaryotic 18S rRNA genes was amplified by using A-528F (5′-GCG GTA ATT CCA GCT CCA A-3′) and B-706R (5′-AAT CCR AGA ATT TCA CCT CT-3′) primer pair (Cheung et al. 2010). The PCR amplification cycles consisted of initial denaturation at 95°C for 5 min, followed by 25 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 60 s, and a final extension at 72°C for 10 min. The purified PCR products were sequenced using an Illumina platform (Illumina Inc., San Diego, CA, USA).

Statistical analysis
The Kruskal-Wallis test was employed to examine the seasonal variation of Giardia and Cryptosporidium in the surface water (intakes of the DWTPs and CUs). Significance was attributed to P values below 0.05. Statistical analyses and visualization were performed using Origin (Pro) 2021, PRIMER v.7.0.21, and R v4.2.2 (https:// www.r-proje ct. org/).

Morphological characteristics of Giardia cysts and Cryptosporidium oocysts using IFA
The positive samples containing Giardia cysts and Cryptosporidium oocysts were easily distinguishable through their apple-green color when stained with an immune-fluorescent stain (FITC). The oval shape cyst wall of the Giardia cysts made them easily recognizable, and each cyst contained 2-4 nuclei that were identifiable by DAPI stain. The size of the detected Giardia cysts ranged from 8-18 × 5-15μm (as shown in Fig. 2). Similarly, the rounded shape oocyst wall of Cryptosporidium oocysts made them easily detectable, and each oocyst contained 4 sporozoites that were visible with DAPI stain. The detected oocysts had a diameter of 4-6μm (Fig. 2).

Occurrence and removal of Giardia and Cryptosporidium in DWTPs and CUs using IFA
The immunofluorescence stain detected Giardia cysts in 11.5% of all collected water samples (both raw and treated), while Cryptosporidium cysts were detected in 18.8% of the  (Table S1). In the inlets of conventional DWTPs (Imbaba and Shubra Alkheymah), the prevalence of Giardia cysts ranged from 8.3 to 33.3%, while it was 16.7% in the inlets of CUs (Mansheyat Alqanater and Niklah). No Giardia cysts were detected in the final treated drinking water of Imbaba and Shubra Alkheymah DWTPs. However, the final treated drinking water of Mansheyat Alqanater and Niklah CUs was contaminated by 8.3% of Giardia cysts (Table 1). Furthermore, higher prevalence rates of Cryptosporidium oocysts were observed in the inlets of both conventional DWTPs (range: 25-41.7%) and CUs (16.67-33.3%).
Cryptosporidium cysts were detected in 16.7% of CUs outlet water samples, while none was found in conventional DWTPs outlet samples (Table 1).

Occurrence and removal of Giardia and Cryptosporidium in DWTPs and CUs using qPCR
The qPCR analysis detected Giardia in 12.5% of all collected water samples (both raw and treated). Additionally, the qPCR analysis identified Cryptosporidium in 20.83% of all collected water (Table S1). More data have been provided in Table S2. The number of positive samples for Giardia was higher in the inlets of Imbaba DWTP (n = 4) compared to other drinking water plants (n = 1 or 2). However, the inlets of Niklah CU and Shubra Alkheymah DWTP had more positive samples for Cryptosporidium (n = 5 for each) compared to Mansheyt Alqanater CU and Imbaba DWTP (n = 3 for each). Overall, it was observed that conventional DWTPs showed higher efficiency in removing Giardia and Cryptosporidium compared to CUs. Both Giardia and Cryptosporidium removal percentages reached 100% in Shubra Alkheymah and Imbaba DWTPs. In contrast, Mansheyat Alqanater and Niklah CUs achieved Cryptosporidium removal percentages of 33.3% and 60%, respectively (Table 2). In the inlets of conventional DWTPs (Imbaba and Shubra Alkheymah), the prevalence of Giardia genes ranged from 8.3 to 33.3%, while it was 16.7% in the inlets of CUs (Mansheyat Alqanater and Niklah). No Giardia genes were detected in the final treated drinking water of Imbaba and Shubra Alkheymah DWTPs. However, a similar occurrence percentage (16.7%) of Giardia genes was found in the final treated drinking water of Mansheyat Alqanater and Niklah CUs (Table 1). Furthermore, higher prevalence rates of Cryptosporidium genes were observed in the inlets of both conventional DWTPs (range: 25-41.7%) and compact units (25-41.7%). Cryptosporidium genes were detected in 16.7% of CUs outlet water samples, while none was found in conventional DWTPs outlet samples ( Table 1).
The counts of Giardia and Cryptosporidium genes in the inlet water samples of conventional DWTPs ranged 0-2.61 GC/10L and 0-2.54 GC/10L, respectively. Giardia and Cryptosporidium were not detected in the outlet water samples of Imbaba and Shubra Alkheymah DWTPs. The maximum concentration of Giardia and Cryptosporidium was 2.2 GC/10L and 2.6 GC/10L, respectively, in the inlet water samples of the CUs, while the maximum concentration of the same parasites in the outlet water samples of the CUs was 1.17 and 2.0 GC/10L, respectively (Fig. 4). No seasonal variations for Giardia and Cryptosporidium (Kruskal-Wallis test: P > 0.05) in surface water samples were observed.

18S rRNA amplicon sequencing
A total of 3447 microeukaryotic ASVs were detected in inlets and outlets of the DWTPs and CUs using 18S rRNA next-generation sequencing. The barplot showed the relative abundance of the top ten microeukaryotic taxa at Rank 3. Several taxa groups (e.g., Ciliophora, Cercozoa) revealed a significant abundance in the inlets and outlets of the DWTPs and CUs. Fungi and Metazoa were the most abundant taxa groups (≥ 13.6%) and (≥ 4.8%) in different DWTPs and CUs stages, respectively. Lower abundant taxa such as Apicomplexa is not shown in Fig. 5. From the lower abundant taxa, Cryptosporidium was detected in the inlet of Mansheyat Alqanater CU (Fig. 6). Several other pathogenic or potentially pathogenic genera (e.g., Blastocystis and Vermamoeba) were detected using 18S rRNA amplicon sequencing.

Discussion
DWTPs employ a combination of physical, chemical, and biological processes to remove contaminants from source water. The effectiveness of these processes in eradicating Cryptosporidium and Giardia, two common waterborne protozoan parasites, relies on several factors, such as the parasite size, treatment method employed, and operational conditions of the plant. Research has shown that Cryptosporidium and Giardia can be effectively removed through conventional DWTPs that include filtration and disinfection steps. However, the removal efficiency varies depending on the specific treatment process and the operational conditions. These findings align with the results of a previous study conducted in Egypt, where the removal percentages of Giardia and Cryptosporidium by conventional DWTPs were also reported as 100% (Ali et al. 2004). In Malaysia, the conventional DWTP demonstrated a removal rate of 92.9% for Giardia and 100% for Cryptosporidium (Richard et al. 2016). Similarly, in Southern Brazil, the DWTP, which encompasses a comprehensive water treatment cycle, achieved a 100% removal rate for both Giardia and Cryptosporidium (Almeida et al. 2015). In a Spanish investigation, conventional DWTPs achieved a 100% removal rate for both Cryptosporidium and Giardia (Carmena et al. 2007). On the contrary, a study conducted in China reported a lower removal percentage (66.7%) for Giardia through conventional DWTPs, while no removal was observed for Cryptosporidium (Kui et al. 2021). Regular monitoring of these rates is crucial to ensure the effectiveness of the treatment processes in eliminating parasites from the water and delivering safe drinking water to consumers. Less information is available on the removal rates of Cryptosporidium and Giardia through CUs for drinking water treatment. CUs are typically designed to treat smaller volumes of water and are commonly utilized in remote or rural areas where conventional water treatment methods may not be accessible (Al-Herrawy and Gad 2017; Ali et al. 2004). In Egypt, the researchers found Cryptosporidium and Giardia in the inlet water samples of the two CUs only (Ali et al. 2004). In the present study, the removal rate of Giardia and Cryptosporidium in the CUs reached up to 60%. Similarly, a study on Spanish CUs with a comparable structure reported a removal rate of 64.3% for Giardia, while Cryptosporidium was not effectively removed by the system (Carmena et al. 2007). The removal rates of Cryptosporidium and Giardia in conventional drinking water treatment plants (DWTPs) can fluctuate depending on several factors, such as changes in the source water quality, variations in the plant operating conditions, and the effectiveness of treatment processes. Compared to conventional DWTPs, some compact units (CUs) may exhibit lower parasite removal rates due to their smaller size or limited treatment capacity.
In the current research, the average prevalence of Cryptosporidium and Giardia in the Nile River (intake water samples) was 33.3% and 20.8%, respectively. Similar findings were reported for Cryptosporidium prevalence in the Nile River, Egypt (33% by direct microscopy) (El-Khayat et al. 2022) and in raw water samples in Iran (30% by IFA) (Mahmoudi et al. 2013). The higher prevalence rate for Giardia in raw water samples collected from the Quindío River basin was reported in Colombia (43.6% by PCR) (Pinto-Duarte et al. 2022), for Cryptosporidium and Giardia in Greece (> 47% by IFA) (Ligda et al. 2020), Giardia cysts and Cryptosporidium oocysts in Canadian rivers (> 63% by IFA and > 50% by PCR) (Prystajecky et al. 2014). However, a lower prevalence of Giardia cysts in Ethiopian rivers (16% by IFA) was recorded (Kifleyohannes and Robertson 2020).
In the present study, the concentration of Giardia cysts in raw water ranged from 0 to 15 cysts/10L, while the Cryptosporidium concentration in raw water ranged from 0 to 21 oocysts/10L. Other studies have reported different concentration ranges for Cryptosporidium and Giardia in raw water samples from various regions. For instance, in Greece, the Cryptosporidium oocyst concentration ranged from 0 to 0.9 oocysts/10L, and the Giardia cyst concentration ranged from 0 to 4.3 cysts/10L (Ligda et al. 2020). In Iran, the Giardia cyst concentration ranged from 1 to 1800 cysts/10L (Mahmoudi et al. 2013), and in Taiwan, the Giardia cyst and Cryptosporidium oocyst concentration in raw water samples ranged from 0.2 to 31.2 cysts/10L and from 0.23 to 80.14 oocysts/10L, respectively (Hsu et al. 1999). In Ethiopia, the Giardia cyst and Cryptosporidium oocyst concentration in raw water samples Fig. 6 Heatmap illustrating the abundance of select rare microeukaryotic genera in the DWTPs and CUs. Color gradient ranges from blue (low abundance) to red (high abundance). Samples were collected from inlet and outlet of CUs (Niklah and Mansheyat Alqanater) and DWTPs (Imbaba and Shubra Alkheyma) ranged from 3 to 22 cysts/10L and from 1 to 3 oocysts/10L, respectively (Kifleyohannes and Robertson 2020). Therefore, it is important to monitor water quality regularly and choose a treatment technology that is appropriate for specific conditions.
In the present study, no significant seasonal variations were observed for Giardia and Cryptosporidium in the surface water. A previous study conducted in Colombia (Escobar et al. 2022) also reported that seasonality did not have a strong impact on the occurrence of Cryptosporidium and Giardia in drinking water systems. In contrast, a study conducted in Egypt (Hamdy et al. 2019) found evidence of seasonality in the prevalence of both Giardia and Cryptosporidium cysts in tap water, with higher levels observed during the summer season. The prevalence and concentration of Cryptosporidium and Giardia in source waters can also vary widely depending on the location, season, sample volume, and detection methods employed. One potential limitation of this study was the use of membrane filters as the concentration method instead of cartridge filtration, which may have influenced the prevalence and concentration of Cryptosporidium and Giardia detected in the water samples. In a previous study by Wohlsen et al. (2004), various filtration methods including Pall Life Sciences Envirochek (EC) standard filtration and Envirochek high-volume (EC-HV) membrane filters, the Millipore flatbed membrane filter, the Sartorius flatbed membrane filter (SMF), and the Filta-Max (FM) depth filter were evaluated for recovery of Cryptosporidium parvum oocysts and Giardia lamblia cysts. The EC-HV membrane filter (EC-HV-R) showed the highest range of recovery rates for both oocysts (36-76%) and cysts (44-72%), followed by the FM depth filter (oocysts: 18-39%; cysts: 30-63%) and SMF (oocysts: 12-19%; cysts: 32-40%) (Wohlsen et al. 2004). However, a contrasting study conducted by Hsu et al. (1999) found that the membrane filtration method exhibited higher recovery rates and detection limits for Giardia and Cryptosporidium compared to the cartridge filtration method. Another potential limitation of the study is the inability to identify the genotypes and species of the parasites. The rare parasitic genus Cryptosporidium was detected in the inlet of Mansheyat Alqanater CU using 18S rRNA amplicon sequencing, and this finding was subsequently confirmed by qPCR. While the qPCR analysis detected Cryptosporidium in the outlet sample, it was not detected through 18S rRNA amplicon sequencing (Table S3). This discrepancy might be attributed to the higher sensitivity of qPCR in detecting low-concentration levels.

Conclusion
The conventional treatment processes, including coagulation, sedimentation, and filtration, have demonstrated greater effectiveness in removing Cryptosporidium and Giardia from drinking water. Specifically, neither Giardia nor Cryptosporidium was detected in conventional DWTPs, indicating the efficiency of these processes in eliminating these parasites from the source water and ensuring the safety of drinking water for consumers. Indeed, no seasonal variations for both parasites were observed. The prevalence of Cryptosporidium oocysts was observed to be higher than Giardia cysts in Nile water, suggesting a potentially higher risk of infection associated with Cryptosporidium, as this parasite can cause gastrointestinal illness even at low doses. The selection of technology for removing Cryptosporidium and Giardia from drinking water should be based on a comprehensive evaluation of the specific context and conditions. The presence of Cryptosporidium and Giardia cysts in CUs outlets does not automatically make this technology less effective, but rather highlights the importance of regular monitoring and maintenance practices. Ultimately, the goal should be to provide safe and reliable drinking water to consumers.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). This study was funded by the Holding Company for Drinking Water and Wastewater, Egypt and performed with technical assistance from Environmental Parasitology Laboratory, Water Pollution Research Department, National Research Centre, Egypt.

Data availability
The manuscript does not contain any material from third parties, and all the material is owned by the authors, and no permission is required for publication.

Declarations
Ethics approval Not applicable.

Conflict of interest The authors declare no competing interests.
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