Different Detection and Treatment Methods for Entamoeba histolytica and Entamoeba dispar in Water/Wastewater: A Review

Rakib Ahmed Chowdhury^1*, Nwadiuto Esiobu², Daniel E. Meeroff¹, Fred Bloetscher¹

¹Department of Civil, Environmental & Geomatics Engineering, Florida Atlantic University, Boca Raton, FL, USA.
²Department of Biological Sciences, Florida Atlantic University, Boca Raton, FL, USA.

DOI: 10.4236/jep.2022.131008                

Abstract

Entamoeba histolytica is an anaerobic parasitic protozoan and well known as a human pathogen, while its close relative, Entamoeba dispar, also possesses similar characteristics as an infectious agent. These microorganisms are generally transmitted in fecal-contaminated water. However, E. dispar present in industrial wastewater is also capable of creating biofilms that can cause adverse impacts in piping networks. Therefore, it is important to detect both of these protozoan species in water and to find a cost-effective technique for inactivation or management control. This review article summarizes the available detection methods in water and wastewater matrices along with feasible disinfection techniques.

Keywords

Entamoeba histolytica, Entamoeba dispar, Detection Methods, Water and Wastewater Treatment

1. Introduction

There are six species of the genus Entamoeba that are found in the human intestinal lumen including Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba poleki, Entamoeba coli, and Entamoeba hartmanni [1]. Among the six species, Entamoeba histolytica is considered to be a human pathogen [2]. Infection leads to the disease known as amebiasis, which is a common cause of death due to parasitic infestation, second only to malaria [3]. Symptoms include diarrhea with cramping, lower abdominal pain, low grade fever, releasing blood and mucus containing stools, and flask-shaped ulcers [4], as shown in Figure 1.

Figure 1. Flask-shaped ulcers developed due to amebiasis [5].

Amebiasis is responsible for about 100,000 deaths each year [6] [7]. The National Institute of Allergy and Infectious Diseases (NIAID) has classified E. histolytica as a category B priority biodefense pathogen [8]. Although humans are the only notable hosts for E. histolytica, it can be a zoonotic parasite. Ai et al. [9] investigated the feces of different animals and found E. histolytica in horses and other Entamoeba species in camels, yaks, sheep, and goats.

Although morphologically identical to Entamoeba histolytica, Entamoeba dispar was until recently considered a non-pathogenic parasite. Several studies [10] [11] [12] have reported intestinal symptoms in patients infected with Entamoeba dispar. In addition, Graffeo et al. [3] mentioned a rare case of enteritis in Italy, a non-endemic country. In addition, Oliveira et al. [13] stated that both Entamoeba histolytica and Entamoeba dispar infect 12% of the world’s population, where the former is responsible for only 1% of the infections. Epidemiological surveys mentioned in Oliveira et al. [13] indicated that most of the asymptomatic infections are caused by Entamoeba dispar.

Moreover, a DNA extraction analysis of diarrheal stool samples in Northwest Ethiopia revealed that about 42.2% of infections were caused by Entamoeba dispar [14]. In the study of Calegar et al. [15], 57.1% of the infected samples contained Entamoeba dispar, 23.8% Entamoeba histolytica, and 14.3% possessed both the parasites. According to Sukprasert et al. [10], one of the major environmental transport systems of E. dispar is via water. Infection of the parasite into the human body is most likely to occur by drinking fecal-contaminated water or ingesting food irrigated with fecal-contaminated water [4]. Particularly in developing countries, drinking water is supplied to residents without proper treatment necessary to inactivate Entamoeba sp. [10].

Entamoeba dispar does not only infect human and non-human hosts, but it is also capable of being a pioneering agent to create biofilms. Meeroff et al. [16] investigated the microbial community structure formed along the inner walls of an industrial wastewater deep injection disposal well at the Solid Waste Authority (SWA) of Palm Beach County, Florida and identified the most abundant microorganism in the biofilm to be Entamoeba dispar (>30%). The formation of the biofilm reduced the injectivity rate by nearly 40% from 1.46 - 1.48 m³/hr/kPa to 0.86 - 1.02 m³/hr/kPa. Currently, there are over 150,000 deep injection well facilities in the United States at an average cost of approximately $8 - 9 million per facility. Reduction of injectivity increases the cost of pumping and may eventually cause catastrophic failure resulting in replacement. Therefore, the objectives of this review article are to 1) Describe the occurrence of Entamoeba species in nature; 2) Review different methods of detection for Entamoeba species, particularly in water/wastewater samples; 3) Discuss disinfection options to control the population of Entamoeba species in water and wastewater matrices.

2. Occurrence in Nature

Entamoeba dispar is found to occur in nature 10 - 14 times more often than Entamoeba histolytica [1] [17]. Ayed et al. [18] indicated that the most common sources of Entamoeba dispar include human feces and consequently, raw sewage and septic tanks. It is also commonly found in cold regions along with tropical and sub-tropical regions containing contaminated wastewater [19]. Table 1 summarizes the occurrence distribution of Entamoeba species in the natural environment.

In addition, Entamoeba species are also documented in Yemen, Northern South Africa, Southwestern China, Bangladesh, Vietnam as well as in Central and South America [35] - [40].

Entamoeba species can also be spread among different environmental media mainly through surface runoff, where it is transported from contaminated soil to surface water [19] [41]. In addition, leakage from underground storage and septic tanks can carry the parasite species to groundwater [42] [43]. Moreover, seepage of contaminated water through subsoil surfaces can transport the Entamoeba species to well water [25]. Survival of Entamoeba species in different environments is highly dependent on temperature. Table 2 summarizes the effects of temperature on the survival of the parasitic cysts. However, in extreme cold or warm conditions such as temperatures below 5˚C or over 40˚C, cysts inactivate rapidly [19].

Table 1. Occurrence of Entamoeba species in natural environments.

Table 2. Effect of temperatures on survival of Entamoeba species in the environment [19].

3. Morphology and Biology

The genus Entamoeba falls in the phylum of Sarcomastigophora and Lobosea class of the protozoan sub-kingdom where its order and family are Amoebida and Endamoebida, respectively. Entamoeba is closely related to other types of parasites such as Rhizopoda and Amoebozoa. Cysts of both Entamoeba histolytica and Entamoeba dispar are morphologically indistinguishable and range in size from 10 - 20 µm (typically 12 - 15 µm) in diameter, while the shape is spherical in bright-field microscopy [44]. Mature cysts are characterized by 4 nuclei, where immature cysts possess only 1 or 2 nuclei [1] [45]. Similar to the cysts, trophozoites of both the species are also identical. In general, the size of a trophozoite is around 10 - 60 µm and contains a single nucleus [44]. The life cycle of Entamoeba species and its relationships with the host is shown in Figure 2.

From the US Centers for Disease Control and Prevention [44], the life cycle of Entamoeba starts when cysts and trophozoites are released to the environment via human feces from infected individuals with diarrhea. However, cysts can be released even in formed stools [44]. In general, cysts are able to survive in an open environment for up to one month [19] before getting mixed with water, soil, crops, etc. from where transmission can occur. On the other hand, trophozoites get destroyed relatively quickly (on the order of days to weeks) once they are released outside of the human body [44].

Ingestion of mature cysts occurs through the drinking of contaminated water and food that travels through the stomach to reach the small intestine. If trophozoites are ingested, they will not be able to survive the gastrointestinal environment. Once in the small intestine, trophozoites are released from the cysts (excystation) and then travel to the large intestine where they multiply by binary fission and eventually produce new cysts, which are passed in feces to continue the life cycle in search of another host [44].

Inside a human host, pathogenic trophozoites inhabit in the gut lumen and once in the colon, pathogenic trophozoites degrade the mucosal layer to bind with epithelial cells [46]. This path is known as commensal colonization. Only in 10% of infections [47] [48], amoebiasis occurs. The mechanisms of the infection of hosts caused by pathogenic trophozoites are summarized in the following flowchart (Figure 3).

Figure 2. Human-parasite relationship for Entamoeba species [44].

4. Laboratory Diagnostic Techniques

Several diagnostic procedures have been reported to detect Entamoeba species in water samples including microscopic analysis, PCR techniques and ELISA. They are discussed briefly as follows.

4.1. Microscopy

Microscopy analysis to identify Entamoeba sp. in feces include wet preparation, concentration, and permanently stained smears [1]. According to Fotedar et al. [1] and Huston et al. [49], microscopic analysis of direct saline mounts is usually conducted on a fresh specimen, but it is an insensitive method (10%). It is recommended by Fotedar et al. [1] to analyze samples within 1 hour of collection to explore motile trophozoites that may carry red blood cells. In general, the concentration method is enough to identify cysts, but the use of permanently stained smears is an essential process to recover and detect Entamoeba species (Fotedar et al.) [1].

Figure 3. Flowchart of the host’s infection mechanisms by pathogenic trophozoites of E. histolytica.

Different types of staining have been used in various studies to detect the parasite species in stool and blood samples. Wright-Giemsa stains and acid-fast stain (modified Ziehl-Neelsen stain) are two commonly used stains. Ezenwa et al. [50] used Giemsa stain to detect E. histolytica from blood samples. The study used diluted Giemsa’s stain (1:10) with pH 7.2 buffer to cover the fixed blood smears for 30 minutes. Then, each slide was bottle dried and air dried by keeping at a vertical position in a dust free environment. Abdel-Hafeez et al. [51] used both acid-fast stain and Giemsa stain to detect E. histolytica from stool samples. In the case of water samples, Al-Khalidy and Jabbar [31] employed modified Ziehl-Neelsen stain for the microscopic analysis.

Fixative processes should be employed to prevent the degradation of the trophozoites of the parasites. Schaudinn’s fluid, merthiolate iodine-formalin, sodium acetate-formalin (SAF) are some of the common fixatives used for the concentration procedure [1]. In general, SAF fixative consists of 5% formalin and 2% acetic acid. Figure 4 and Figure 5 represent microscopic images of Entamoeba histolytica/dispar trophozoites and cysts with different types of staining respectively.

Figure 4. Entamoeba histolytica/dispar trophozoites stained with trichrome [52].

(a) (b) (c) (d) (e) (f)

Figure 5. Photographs taken of Entamoeba histolytica/Entamoeba dispar cysts. (a) Cyst in an unstained concentrated wet mount of stool; (b) Cyst in an unstained concentrated wet mount of stool; (c) Cyst in a concentrated wet mount stained with iodine; (d) Cyst in a concentrated wet mount stained with iodine; (e) Cyst stained with trichrome. Note the chromatoid body with blunt ends (red arrow); (f) Cyst stained with trichrome. Three nuclei are visible in the focal plane (black arrows), and the cyst contains a chromatoid body with typically blunted ends (red arrow) [52].

Al-Nihmi et al. [53] collected treated sewage water to detect the presence of Entamoeba sp. At first, the wastewater specimens were prefiltered to remove coarse particles. Next, the sample was settled for 2 hours to allow sediments and large particles to be removed by gravity. Next, 90% of the supernatant was discarded, and the remaining 10% sediments were centrifuged at 1000 G for 15 minutes before the pellets were suspended in 10 mL buffer solution (pH = 4.5). Next, the prepared solution was centrifuged at 1000 G for 1 minute. The pellets were suspended in 6 mL of 10% formol water followed by addition of 4 mL dimethyl ether and vortexing to obtain a uniform mixture. The whole mixture was centrifuged at 1000 G for 1 minute. The supernatant was discarded, and formed pellets were suspended in saline. A small portion of the pellets were transferred to a slide for the microscopic analysis. The study found an average of 22.2% of the tested samples as positive for E. histolytica cysts or trophozoites over a three-month surveillance period.

In Tunisia, Sabbahi et al. [34] analyzed 5 L of raw sewage or partially treated wastewater and 10 L of treated wastewater with secondary biological treatment for the presence of Entamoeba sp. Samples were settled to remove sediments at ambient temperature, and the supernatant was removed using a pump. Then, the prepared sediment was centrifuged in 15 - 50 mL centrifuge tubes with lids at 1000 G for 15 minutes. Next, the pellets were suspended in equal volumes (double the amount of the pellet) of acetoacetic acid buffer (pH = 4.5) followed by the addition of ethyl acetate. After mixing the sample for 10 minutes, it was centrifuged at 1000 G for 15 minutes, resulting in the formation of three distinct layers (e.g. a black layer on top, a turbid layer in the middle, and a sediment layer at the bottom). After that, the suspended layer was resuspended in five volumes of zinc sulfate solution with a specific gravity of 1.18 (density 33%) and mixed thoroughly. Then, 5.0 µL of resuspended material is placed on a slide for microscopic detection magnifying by ×100 and ×400. The number of protozoan cysts per liter is calculated using the following mathematical expression [34]:

$N=AX/PV$ (1)

where, N = number of cysts per liter, A = number of cysts counted in microscopy analysis, P = volume used for the microscopy examination, and V = initial sample volume.

In another study conducted in Germany [45], raw sewage and wastewater samples were passed through a 0.3 mm sieve for the removal of coarse solids. Then, the sample was centrifuged at 4500 G for 30 minutes followed by filtration using 0.22 µm nitrate cellulose membranes. After filtration, the sample was stained and examined in light microscopy for the identification of Entamoeba species. Microscopy techniques for identifying Entamoeba are not as reliable as cultures and isoenzyme analysis, as the sensitivity of the method is very poor (60%) [1]. It is very difficult to differentiate Entamoeba histolytica and Entamoeba dispar using microscopy, as these two species are morphologically identical to each other [54] [55] [56]. Pillai et al. [57] reported that the accuracy of Entamoeba identification was only 9.5% compared to that of other methods such as the PCR and ELISA. Microscopy is only used to identify Entamoeba species, while Polymerase Chain Reaction (PCR) is usually followed to differentiate Entamoeba histolytica and Entamoeba dispar [58].

4.2. PCR Techniques

PCR is a molecular technique to detect the presence of target microorganism by increasing the number of copies of DNA. The approach requires DNA templates, primer pairs, DNA polymerases and dNTPs to increase the copies of the nucleic acids by denaturation, annealing, and extensions. The steps are controlled by a thermocycler, which varies the temperature for specific times.

There are three types of PCR tests available: conventional PCR (C-PCR), nested PCR (N-PCR) and real time PCR assay (RT-PCR). Kim et al. [59] discussed the comparison of the three types of PCR assays for Vibrio vulnificus. In C-PCR, only a single set of primers are used. N-PCR is a modified PCR test that employs two sets of primers to increase sensitivity. For N-PCR, two successive PCR tests are conducted, where the amplified products from the first reaction are used as templates for the second reaction [60]. In the case of RT-PCR, fluorescent dyes are used for measuring amplification to evaluate the number of DNA copies and can be conducted by using 18 S rRNA as the PCR target [61]. To conduct the PCR analysis, a reference sequence is needed for each of the target microorganism. In most of the cases, the PCR target sequences are derived from the NCBI GeneBank database. For E. histolytica and E. dispar, different types of reference sequences were used in different studies. A brief summary of the reference sequences employed in the previous studies are provided in Table 3.

For the detection of Entamoeba species present in wastewater samples, it is required to purify/concentrate the specimens prior to nucleic acid extraction. Table 4 describes several purification/concentration procedures and DNA/RNA extraction kits employed in previous studies to recover nucleic acids from E. histolytica and E. dispar in water and wastewater samples.

The next step is to design the primer sequences for the target genomes. A number of online software packages are available to design the primers including Primer Blast from NCBI, Primer 3. Primerselect, Dansis Max, NetPrimer,

Table 3. Summary of the reference sequences used in the previous studies.

Table 4. Purification/concentration procedures and nucleic acid extraction kits used for detection of Entamoeba species.

Array designer 2, Fast PCR, Oligo 7, Prime designer 4, Gprime and others. The ideal length of a primer is between 18 - 24 bp. The primer works as a pair, and the differences between annealing temperatures of primers in a pair should be less than 3˚C. Several designed primer pairs, selected genome types, amplifying product sizes, preparation procedures for amplification and cycles used in previous studies are described in Table 5. Some primers were designed to be species-specific for Entamoeba histolytica and Entamoeba dispar separately; where in other cases, the primers were designed for both (genus-specific).

4.3. Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA is another technique to identify the presence of the Entamoeba species in environmental water samples or from stool or blood samples. For Entamoeba species, ELISA is generally conducted to detect serum IgG antibody against the microorganisms [70] as it is demonstrated to have improved sensitivity [71]. A number of commercially available test kits have been used including Entamoeba histolytica II test kits, RIDASCREEN E. histolytica IgG, Entamoeba Celisa Path and others [55] [70] [72] [73]. The US Environmental Protection Agency (USEPA) established standard procedures to detect Cryptosporidium parvum and Giardia lamblia in wastewater by using Immuno-Magnetic Separation (IMS) (USEPA Method 1623). Although, it is a popular detection method for Entamoeba species, it has lower sensitivity than that of the PCR assays. In general,

Table 5. Designed primer pairs, selected genome types, amplifying product size, amplification procedure and followed cycles from previous studies.

PCR is 100 times more sensitive than ELISA [1]. Gonin and Trudel [55] found that microscopic analysis possessed higher sensitivity than that of the ELISA particularly when few numbers of the microorganism was present. However, it is less expensive and relatively easier to detect the protozoan species with ELISA compared to PCR analysis [74].

5. Treatment to Inactivate Entamoeba Species

5.1. Physical Treatment Processes

Since Entamoeba species are highly resistant to common disinfectants and both trophozoites and cysts are relatively larger than most of the other common waterborne microorganisms, physical treatment processes can effectively remove the protozoan species. In the case of primary sedimentation, removal of protozoa (Giardia lamblia) was 0.11 log₁₀ [75]. The removal rates of the Entamoeba cysts were reported as 0.49 log₁₀ using an advanced primary treatment process [76]. Rapid sand filtration was found to remove 1.0 log₁₀ at filtration rates less than 2.4 m/hr [19]. In addition, Bitton, Jimenez et al. and Leong reported [76] [77] [78] that further inactivation (2.0 log₁₀) can be accomplished by the addition of a coagulant. However, it is recommended to conduct flocculation before primary treatment [75]. A report published by the US Army [79] cited by Schaefer et al. [80] revealed 98.5% and 99.8% removal of Entamoeba histolytica by coagulation with alum and soda ash, respectively followed by sedimentation. Jimenez et al. [81] used a Microsand (0.1 - 1.0 mm DIA) filter along with aluminum sulfate (Al₂(SO₄)₃) (40 - 60 mg/L) as a coagulant.

Shukla et al. [82] used chitosan oligosaccharide-coated iron oxide nanoparticles to remove E. histolytica while employing an external magnetic field. The nanoparticles were synthesized and incubated in a contaminated water sample containing known numbers of protozoa cysts. The concentration of the nanoparticles and incubation period were varied at a pH of 7.0 and a temperature of 37˚C. The protozoan cysts were attached to the synthesized nanoparticle material during incubation and removed using a magnetic separator. The maximum removal percentage (86%) was obtained at a 4 mg/mL of nanoparticle concentration for an incubation time of 35 minutes.

5.2. Radiation

Mtapuri-Zinyowera et al. [83] investigated the use of solar radiation to inactivate Entamoeba species. Cysts were inactivated efficiently at 50˚C due to the synergistic effect of solar radiation and heat, with total eradication achieved at 56˚C. Mohamed et al. [84] also measured the efficiency of solar energy to destroy Entamoeba species with one-sided blackened bottles vertically exposed to the sun for 7 hours at an ambient temperature of 40˚C and allowing the temperature to rise to 50˚C - 60˚C. They found complete elimination at a temperature above 56˚C.

Maya et al. [85] investigated ultraviolet (UV) light to inactivate amphizoic amoebae present in water samples. The researchers used Acanthamoeba culbertsoni and Acanthamoeba species as the target microorganisms and found that a high UV dose of 173 mW∙s/cm² is needed for an effective contact time period of 1200 sec to achieve complete inactivation of the tested species. In addition, the study concluded that dose of 60 mW∙s/cm² of UV light is required to achieve a 2.0-log inactivation. However, further investigations are needed to reveal the dose and effective contact time to inactivate Entamoeba species.

Ryu et al. [86] found that using UV/TiO₂ enhanced inactivation by reducing the dosage required by 56% compared to UV alone for oocyst removal. Several studies [86] [87] [88] investigated UV/TiO₂ photocatalytic inactivation of protozoan species. A complete inactivation of Giardia intestinalis can be obtained after 30 minutes contact in a UV/TiO₂ system [87]. Inactivation rates for Cryptosporidium parvum oocysts were 1.3, 2.6 and 3.3 log₁₀ at UV dosages of 2.7, 8.0 and 40.0 mJ/cm², respectively [86]. Therefore, this can also be an emerging technique to inactive the Entamoeba species.

5.3. Disinfection Processes

Although, chlorine is used worldwide for disinfection, Entamoeba species are particularly resistant to typical dosages [19]. However, there have been several studies (presented in Table 6) where the required CT values under different pH and temperatures were investigated. In general, chlorine disinfection is more efficient at an acidic pH (<7.0) when more of the chemical is in the hypochlorous acid (HOCl) form. The effectiveness of chlorine as a disinfectant is highly temperature and pH dependent. The most powerful disinfectant is gaseous Cl₂, followed by hypochlorous solution and chloramines, which are the least effective [89].

Bromine can be a more effective disinfectant against Entamoeba species compared to chlorine. Stringer et al. [92] cited by WHO [94] examined that a bromine dose of 1.5 - 4.0 mg/L with a contact time of 10 minutes can achieve

Table 6. Different concentration of Cl₂ dosages and suitable working temperatures and pH.

3.0 log₁₀ inactivation of E. histolytica at a pH and temperature of 4.0˚C - 10.0˚C and 4˚C - 10˚C, respectively. Like chlorine, bromine also works best at a pH of 6.0 - 7.0 since the chemical stays in the hypobromous acid (HOBr) form. Liquid bromine can also be used for disinfection. However, it is recommended to use bromine stick (an organic substance with a mixture of bromine, chlorine and dimethyl hydantoin which is available in tablets or cartridges) instead of liquid bromine because there are risks associated with metal reactivity, and it is corrosive. Bromine is not suitable to use for disinfection of drinking water as it imparts a medicine-like taste and should only be employed in emergency cases [92]. However, a bromine concentration higher than 0.5 mg/L causes eye and mucous membrane irritation [95].

Chang [96] cited by WHO [97] mentioned that elemental iodine (I₂) can be 2 to 3 times more effective for Entamoeba disinfection particularly at a pH range of 5 - 7. In addition, I₂ has greater penetration capability than that of HIO, which makes it an effective disinfectant to use against biofilms in distribution systems, deep injection wells and other facilities. However, further research is required to ensure its effectiveness to penetrate and inactivate Entamoeba colonies formed in hydraulic pipelines.

There have also been limited studies regarding the use of ozone to disinfect Entamoeba species. Due to the greater oxidant potential, ozone is considered highly toxic against waterborne microorganisms [98]. The effluent water samples from the Shahid Beheshti treatment plant in Iran where ozone was being used as the disinfectant were tested for the waterborne parasites, and no living protozoan species were found [98]. This indicates that ozone disinfection can be very effective against E. histolytica and E. dispar. The study of Newton and Jones [99] cited by National Research Council [89] stated that 98% to over 99% inactivation of E. histolytica suspended in water samples can be achieved by a CT value as low as 0.15 mg·min/L. The study also revealed that the disinfection technique provided similar removal efficiency for temperature and pH ranging from 10˚C - 30˚C and 6.5˚C - 8.0˚C, respectively.

Several other disinfection techniques to inactivate protozoa have been reported with mixed results including primary sedimentation, trickling filter with sludge digestion and drying, and oxidation ditch with sedimentation with removal rates of 0.05 to 0.3 log₁₀ [19] [100]. These studies also stated that the waste stabilization pond technique can achieve removal higher than 2.0-log₁₀ with a minimum retention time of 25 days.

6. Conclusions and Recommendations

Both E. histolytica and E. dispar are most commonly found in human feces and as a result, enter the environment via septic tanks and partially treated wastewater releases, especially in developing and tropical weather countries. Although the existence of Entamoeba species depends on temperature, the parasites start to become inactivate quickly over 40˚C and ultimately, become completely inactivated at a temperature above 45˚C. The relationship of the protozoan species with its host and infection mechanisms has been well established. The primary media of transferring Entamoeba species within the hosts’ body are via ingestion of fecal contaminated water and food.

For the detection of the E. histolytica and E. dispar in water and wastewater samples, three techniques were reviewed, namely microscopic analysis, ELISA test and PCR assay. The microscopic test has very low sensitivity, and it is also almost impossible to distinguish the cysts of E. histolytica and E. dispar, as they are morphologically similar. On the other hand, ELISA test is easier to conduct compared to PCR analysis and is also a cost-effective approach. Although it showed lower sensitivity than that of the microscopic analysis in one of the studies, researchers found it to be a more suitable option to detect and differentiate the species. However, there have been limited studies conducted on Entamoeba detection in water or wastewater using ELISA. On the other hand, WHO endorsed the PCR assay to be the most effective approach to detect and differentiate the protozoa species, and the test also possesses a high sensitivity.

A number of studies have already been conducted to propose a suitable and cost-effective approach to inactivate Entamoeba species. Since the parasite species are highly resistant to the normal dosages of chlorine and the size of its cysts is also comparatively larger, filtration can be effective. Currently, available filtration systems are capable of removing 98.5% to 100% of both species from water samples. Addition of coagulants such as alum and soda ash followed by sedimentation or filtration processes increase the removal efficiency. Solar radiation in conjunction with heat can also be a cost-effective approach to inactivate Entamoeba, particularly at temperatures above 50˚C.

Although normal chlorine dosages cannot penetrate and disinfect cysts of E. histolytica and E. dispar, previous studies showed that CT values ranging from 20 to 120 mg·min/L are capable of inactivating 99% to 99.9% of the protozoa present depending on temperature and pH. Several other investigations were conducted to reveal the effectiveness of using bromine as a disinfectant and found that it is more efficient than chlorine, especially at a low temperature ranging from 4˚C - 10˚C. However, bromine creates taste and odor issues while treating for drinking water and hence, it is recommended to use only in emergency cases. Besides chlorine and bromine, iodine can also be another effective disinfectant, but more investigations are required to reveal its suitability.

All the disinfection procedures presented in this review are mainly focused on the inactivation of Entamoeba histolytica. There have been very limited studies about disinfection techniques to specifically remove Entamoeba dispar. In addition, the effectiveness of electron beam technique has never been evaluated for the Entamoeba species. Rawat and Sarma [101] revealed that even 1 kGy of electron beam dose may result in complete inactivation of coliforms, Salmonella-Shigella, E. coli and almost 3.0 log₁₀ removal of bacterial species. Hence, it can be an emerging and efficient technique to treat Entamoeba species. Moreover, investigations are needed to reveal the most suitable approaches to inactivate E. dispar in formed biofilms in pipe networks, which is a case presented in the study of Meeroff et al. [16]. Although Meeroff et al. [16] mentioned several techniques and conditions to penetrate and inactivate biofilm produced by other microorganisms such as Pseudomonas and Giardia lamblia, the recommended inactivation conditions for Entamoeba dispar still remain to be investigated.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

References

[1]	Fotedar, R., Stark, D., Beebe, N., Marriott, D., Ellis, J. and Harkness, J. (2007) PCR Detection of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii in Stool Samples from Sydney, Australia. Journal of Clinical Microbiology, 45, 1035-1037. https://doi.org/10.1128/JCM.02144-06
[2]	Nespola, B., Betz, V., Brunet, J., Gagnard, J.C., Krummel, Y., Hansmann, Y., et al. (2015) First Case of Amebic Liver Abscess 22 Years after the First Occurrence. Parasite, 22, Article No. 20. https://doi.org/10.1051/parasite/2015020
[3]	Graffeo, R., Archibusacci, C.M., Soldini, S., Romano, L. and Masucci, L. (2014) Entamoeba dispar: A Rare Case of Enteritis in a Patient Living in a Nonendemic Area. Case Reports in Gastrointestinal Medicine, 2014, Article ID: 498058. https://doi.org/10.1155/2014/498058
[4]	WHO (World Health Organization) (2008) Guidelines for Drinking Water Quality. 3rd Edition, World Health Organization, Geneva.
[5]	Shirley, D.T., Farr, L., Watanabe, K. and Moonah, S. (2018) A Review of the Global Burden, New Diagnostics, and Current Therapeutics for Amebiasis. Open Forum of Infectious Diseases, 5, Article No. ofy161. https://doi.org/10.1093/ofid/ofy161
[6]	Pal, M. (2020) Amoebiasis: An Important Foodborne Disease of Global Public Health Concern. Archives of Nutrition and Public Health, 2, 1-3.
[7]	Matar, N.K. and Neamah, S.R., (2021) Isolation and Diagnosis of Entamoeba histolytica and Giardia lamblia from Children Infected with Diarrheal at Mohammed AL-Moussawi Hospital in Dhi-Qar Province. International Journal of Science, Engineering and Management, 6, 10-13.
[8]	Cui, Z., Li, J., Chen, Y. and Zhang, L. (2019) Molecular Epidemiology, Evolution, and Phylogeny of Entamoeba spp. Infection, Genetics and Evolution, 75, Article ID: 104018. https://doi.org/10.1016/j.meegid.2019.104018
[9]	Ai, S., Zhang, Z., Wang, X., Zhang, Q., Yin, W. and Duan, Z. (2021) The First Survey and Molecular Identification of Entamoeba spp. in Farm Animals on Qinghai-Tibetan Plateau of China. Comparative Immunology, Microbiology and Infectious Diseases, 75, Article ID: 101607. https://doi.org/10.1016/j.cimid.2020.101607
[10]	Sukprasert, S., Rattapraset, P., Hamzah, Z., Shipin, O. and Chavalistshewinkoon-Pemitr, P. (2008) PCR Detection of Entamoeba spp. from Surface and Wastewater Samples Using Genus-Specific Primers. Southeast Asian Journal of Tropical Medicine and Public Health, 39, 6-9.
[11]	Khairnar, K. and Parija, S.C. (2007) A Novel Nested Multiplex Polymerase Chain Reaction (PCR) Assay for Differential Detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in Stool Samples. BMC Microbiology, 7, Article No. 47. https://doi.org/10.1186/1471-2180-7-47
[12]	Ali, I.K.M., Hossain, M.B., Roy, S., Ayeh-Kumi, P.F., Petri Jr., W.A., Haque, R. and Clark, C.G. (2003) Entamoeba moshkovskii Infections in Children, Bangladesh. Emerging Infectious Diseases, 9, 580-584. https://doi.org/10.3201/eid0905.020548
[13]	Oliveira, F.M.S., Neumann, E., Gomes, M.A. and Caliari, M.V. (2015) Entamoeba dispar: Could It Be Pathogenic. Tropical Parasitology, 5, 9-14. https://doi.org/10.4103/2229-5070.149887
[14]	Yimer, M., Zenebe, Y., Mulu, W., Abera, B. and Sauger, J.S. (2016) Molecular Prevalence of Entamoeba histolytica/dispar Infection among Patients Attending Four Health Centres in North-West Ethiopia. Tropical Doctor, 47, 11-15. https://doi.org/10.1177/0049475515627236
[15]	Calegar, D.A., Nunes, B.C., Monteiro, K.J.L., dos Santos, J.P., Toma, H.K., Gomes, T.F., et al. (2016) Frequency and Molecular Characterization of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, and Entamoeba hartmanni in the Context of Water Scarcity in Northeastern Brazil. Memorias do Instituto Oswaldo Cruz, 111, 114-119. https://doi.org/10.1590/0074-02760150383
[16]	Meeroff, D.E., Shaha, B., Bloetscher, F., Esiobu, N., Mercer, B., McCquordale, D., Kari, R. and Benett, M. (2019) Characterization of Biofilms and Mineralogical Scale in Underground Injection Well Disposal of Landfill Leachate and Industrial Wastewater Systems. Journal of Geoscience and Environment Protection, 7, 69-91. https://doi.org/10.4236/gep.2019.711006
[17]	Nesbitt, R.A., Mosha, F.W., Katki, H.A., Ashraf, M., Assenga, C. and Lee, C.M. (2004) Amebiasis and Comparison of Microscopy to ELISA Technique in Detection of Entamoeba histolytica and Entamoeba dispar. Journal of the National Medical Association, 96, 671-677.
[18]	Ayed, L.B., Schijven, J., Alouini, Z., Jemil, M. and Sabbahi, S. (2009) Presence of Parasitic Protozoa and Helminth in Sewage and Efficiency of Sewage Treatment in Tunisia. Parasitology Research, 105, Article No. 393. https://doi.org/10.1007/s00436-009-1396-y
[19]	Ben Ayed, L. and Sabbahi, S. (2017) Entamoeba histolytica. In: Rose, J.B. and Jiménez-Cisneros, B., Eds., Water and Sanitation for the 21st Century: Health and Microbiological Aspects of Excreta and Wastewater Management (Global Water Pathogen Project). (R. Fayer and W. Jakubowski (eds), Part 3: Specific Excreted Pathogens: Environmental and Epidemiology Aspects—Section 3: Protists), Michigan State University, E.Lansing, MI, UNESCO. https://doi.org/10.14321/waterpathogens.34
[20]	Saleh, F.E.R., Gad, M.A., Ashour, A.A., Soliman, M.I., El-Senousy, W.M. and Al-Herrawy (2018) Molecular Detection of Entamoeba histolytica in Fresh Vegetables and Irrigation. Egyptian Journal of Aquatic Biology and Fisheries, 22, 551-561. https://doi.org/10.21608/ejabf.2019.24756
[21]	Moreno, Y., Moreno-Mesonero, L., Amoros, I., Perez, R., Morillo, J.A. and Alonso, J.L. (2018) Multiple Identification of Most Important Waterborne Protozoa in Surface Water Used for Irrigation Purposes by 18S rRNA Amplicon-Based Metagenomics. International Journal of Hygiene and Environmental Health, 221, 102-111. https://doi.org/10.1016/j.ijheh.2017.10.008
[22]	Mahmoudi, M.R., Nazemalhosseini-Mojarad, E. and Karanis, P. (2015) Genotyping of Giardia lamblia and Entamoeba spp. from River Waters in Iran. Parasitology Research, 114, 4565-4570. https://doi.org/10.1007/s00436-015-4702-x
[23]	Elfadaly, H.A., Hassanain, N.A., Hassanain, M.A., Barakat, A.M. and Shaapan, R.M. (2018) Evaluation of Primitive Ground Water Supplies as a Risk Factor for the Development of Major Waterborne Zoonosis in Egyptian Children Living in Rural Areas. Journal of Infection and Public Health, 11, 203-208. https://doi.org/10.1016/j.jiph.2017.07.025
[24]	Bishop, H.G. and Inabo, H.I. (2015) Incidence of Entamoeba Histolytica in Well Water in Samaru-Zaria, Nigeria. International Journal of Scientific Research in Environmental Sciences, 3, 296-300.
[25]	Chen, K.T., Chen, C.J. and Chiu, J.P. (2001) A School Waterborne Outbreak Involving both Shigella sonnei and Entamoeba histolytica. Journal of Environmental Health, 64, 9-13
[26]	Walters, H.A., Welter, B.H., Sullivan Jr., W.J. and Temesvari, L.A. (2019) Phosphorylation of Eukaryotic Initiation Factor-2α in Response to Endoplasmic Reticulum and Nitrosative Stress in the Human Protozoan Parasite, Entamoeba histolytica. Molecular and Biochemical Parasitology, 234, Article ID: 111223. https://doi.org/10.1016/j.molbiopara.2019.111223
[27]	Matsubayashi, M., Matsuura, Y., Nukata, S., Daizi, Y., Shibahara, T. and Teramoto, I. (2017) First Detection and Molecular Identification of Entamoeba bovis from Japanese Cattle. Parasitology Research, 117, 339-342. https://doi.org/10.1007/s00436-017-5689-2
[28]	Ogbolu, D.O., Alli, O.A., Amoo, A.O., Ilozavbie, G.W. and Olusoga-Ogbolu, F.F. (2011) High-Level Parasitic Contamination of Soil Sampled in Ibadan Metropolis. African Journal of Medicine and Medical Sciences, 40, 321-325.
[29]	Ramos, E.L.P., Gomez-Hernandez, C., Queiroz, L.G., Moura, F.M., Nogueira, N.P., Ferreira, G.L.S. and Rezende-Oliveira, K. (2020) Parasite Detection in Sand from Bays on the North Coast of São Paulo State, Brazil. Journal of Tropical Pathology, 49, 191-205. https://doi.org/10.5216/rpt.v49i3.63783
[30]	Dalzochio, T., Araldi, J.A. and Stuani, L. (2020) Scenario of the Prevalence of Parasites in Soil/Sand Samples in Brazil: Systematic Analysis. Saúde e meio ambiente: Revista Interdisciplinary, 9, 233-247. https://doi.org/10.24302/sma.v9i0.2708
[31]	Al-khalidy K.A.H. and Jabbar, H.K. (2020) Detection of Some Intestinal Parasites in the Wastewater of Some Health Centers (Popular Clinics) in Some Areas of Al-Diwaniyah City, Iraq. Eurasian Journal of Biosciences, 14, 2115-2121.
[32]	Shankar, P., Mishra, J., Bharti, V., Parashar, D. and Singh, S. (2019) Multiplex PCR Assay for Simultaneous Detection and Differentiation of Entamoeba histolytica, Giardia lamblia, and Salmonella spp. in the Municipality-Supplied Drinking Water. Journal of Laboratory Physicians, 11, 275-280. https://doi.org/10.4103/JLP.JLP_66_18
[33]	Ghasemi, E., Rahdar, M. and Rostami, M. (2015) Prevalence of Entamoeba histolytica/dispar in Drinking Water in the City of Shush, Khuzestan Province in 2011. International Journal of Current Microbiology and Applied Sciences, 4, 582-588.
[34]	Sabbahi, S., Trad, M., Ayed, L.B. and Marzougui, N. (2018) Occurrence of Intestinal Parasites in Sewage Samples and Efficiency of Wastewater Treatment Systems in Tunisia. Water Quality Research Journal, 53, 86-101. https://doi.org/10.2166/wqrj.2018.033
[35]	Pu, L.H.., Li, Z.., Wu, J., Zhang, Y., Chen, J., et al. (2020) Prevalence, Molecular Epidemiology and Zoonotic Risk of Entamoeba spp. from Experimental Macaques in Yunnan Province, Southwestern China. Parasitology Research, 119, 2733-2740. https://doi.org/10.1007/s00436-020-06762-9
[36]	Samie, A., Mahlaule, L., Mbati, P., Nozaki, T. and ElBakri, A. (2020) Prevalence and Distribution of Entamoeba Species in a Rural Community in Northern South Africa. Food and Waterborne Parasitology, 18, Article ID: e00076. https://doi.org/10.1016/j.fawpar.2020.e00076
[37]	Al-Areeqi, M.A., Sady, H., Al-Mekhlafi, H.M., Anuar, T.S., Al-Adhroey, A.H., Atroosh, W.M., et al. (2017) First Molecular Epidemiology of Entamoeba histolytica, E. dispar and E. moshkovskii Infections in Yemen: Different Species-Specific Associated Risk Factors. Tropical Medicine and International Health, 22, 493-504. https://doi.org/10.1111/tmi.12848
[38]	El-Dib, N.A. (2017) Entamoeba histolytica: An Overview. Current Tropical Medicine Report, 4, 11-20. https://doi.org/10.1007/s40475-017-0100-z
[39]	Lin, A., Ercumen, A., Benjamin-Chung, J., Arnold, B.F., Das, S., Haque, R., et al. (2018) Effects of Water, Sanitation, Handwashing, and Nutritional Interventions on Child Enteric Protozoan Infections in Rural Bangladesh: A Cluster-Randomized Controlled Trial. Clinical Infectious Diseases, 67, 1515-1522. https://doi.org/10.1093/cid/ciy320
[40]	Duc, P.P., Nguyen-Viet, H., Hattendorf, J., Zinsstag, J., Cam, P.D. and Odermatt, P. (2011) Risk Factors for Entamoeba histolytica Infection in an Agricultural Community in Hanam Province, Vietnam. Parasites & Vectors, 4, Article No. 102. https://doi.org/10.1186/1756-3305-4-102
[41]	Bordinhão, P. and Costa, L. (2006) Areia contaminada das praias da Guanabara Contaminated sand of the Beaches of Guanabara. Bol Comissão Meio Ambiente Assembléia Legislativa Est Rio de Janeiro.
[42]	Ejike, B.U., Ohaeri, C.C. and Amaechi, E.C. (2021) Parasitic Contamination of Local Drinking Water Sourcesin Aba Metropolis, Abia State, Nigeria. SQU Journal of Science, 26, 1-7. https://doi.org/10.53539/squjs.vol26iss1pp1-7
[43]	Abusheishaa, G.A., O.Hafez, A. and Ahmed, H.M. (2021) Efficacy of Triage Parasite Panel in Diagnosis of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum Antigens in Symptomatic Children Stool Specimens. Egyptian Veterinary Medical Society of Parasitology Journal, 17, 84-91. https://doi.org/10.21608/evmspj.2021.192801
[44]	Centers for Diseases Control and Prevention (2015) Pathogen & Environment. https://www.cdc.gov/parasites/amebiasis/pathogen.html
[45]	Ajonina, C., Buzie, C., Moller, J. and Otterpohl, R. (2018) The Detection of Entamoeba histolytica and Toxoplasma gondii in Wastewater. Journal of Toxicology and Environmental Health, Part A, 81, 1-5. https://doi.org/10.1080/15287394.2017.1392399
[46]	Wilson, I.W., Weedall, G.D. and Hall, N. (2012) Host-Parasite Interactions in Entamoeba histolytica and Entamoeba dispar: What Have We Learned from Their Genomes? Parasite Immunology, 34, 90-99. https://doi.org/10.1111/j.1365-3024.2011.01325.x
[47]	Nagaraja, S. and Ankri, S. (2019) Target Identification and Intervention Strategies against Amebiasis. Drug Resistance Update, 44, 1-14. https://doi.org/10.1016/j.drup.2019.04.003
[48]	Li, J., Cui, Z., Li, X. and Zhang, L. (2021) Review of Zoonotic Amebiasis: Epidemiology, Clinical Signs, Diagnosis, Treatment, Prevention and Control. Research in Veterinary Science, 136, 174-181. https://doi.org/10.1016/j.rvsc.2021.02.021
[49]	Huston, C.D., Haque, R. and Petri Jr., W.A. (1999) Molecular-Based Diagnosis of Entamoeba histolytica Infection. Expert Review in Molecular Medicine, 1, 1-11. https://doi.org/10.1017/S1462399499000599
[50]	Ezenwa, C.M., Ukaga, C.N., Emukah, E., Nnagbo, P.A., Obasi, C.C., Nwachukwu, I.O., et al. (2018) Age Related Co-Infection of Malaria, Bacteremia, and Intestinal Parasites in Primary School Children in Owerri, Imo State, Nigeria. International Journal of Innovative Science, Engineering & Technology, 5, 77-83.
[51]	Abdel-Hafeez, E.H., Ahmad, A.K., Ali, B.A. and Moslam, F.A. (2012) Opportunistic Parasites among Immunosuppressed Children in Minia District, Egypt. Korean Journal of Parasitology, 50, 57-62. https://doi.org/10.3347/kjp.2012.50.1.57
[52]	Centers for Diseases Control and Prevention (2019) Amebiasis. https://www.cdc.gov/dpdx/amebiasis/index.html
[53]	Al-Nihmi, F.M., Salih, A.A., Qazzan, J., Radman, B., Al-Woree, W., Belal, S., et al. (2020) Detection of Pathogenic Waterborne Parasites in Treated Wastewater of Rada’a City-Yemen. Journal of Scientific Research in Medical and Biological Sciences, 1, 30-39. https://doi.org/10.47631/jsrmbs.v1i1.23
[54]	Davis, P.H., Chen, M., Zhang, X., Clark, C.G., Townsend, R.R. and Stanley Jr., S.L. (2009) Proteomic Comparison of Entamoeba histolytica and Entamoeba dispar and the Role of E. histolytica Alcohol Dehydrogenase 3 in Virulence. PLoS Neglected Tropical Diseases, 3, Article No. e415. https://doi.org/10.1371/journal.pntd.0000415
[55]	Gonin, P. and Trudel, L. (2003) Detection and Differentiation of Entamoeba histolytica and Entamoeba dispar Isolates in Clinical Samples by PCR and Enzyme-Linked Immunosorbent Assay. Journal of Clinical Microbiology, 41, 237-241. https://doi.org/10.1128/JCM.41.1.237-241.2003
[56]	Haque, R., Ali, I.K.M., Akther, S. and Petri Jr., W.A. (1998) Comparison of PCR, Isoenzyme Analysis and Antigen Detection for Diagnosis of Entamoeba histolytica Infection. Journal of Clinical Microbiology, 36, 449-452. https://doi.org/10.1128/JCM.36.2.449-452.1998
[57]	Pillai, D.R., Keystone, J.S., Sheppard, D.C., MacLean, J.D., MacPherson, D.W., Kain, K.C. (1999) Entamoeba histolytica and Entamoeba dispar: Epidemiology and Comparison of Diagnostic Methods in a Setting of Non-Endemicity. Clinical Infectious Diseases, 29, 1315-1318. https://doi.org/10.1086/313433
[58]	Ngui, R., Angal, L., Fakhrurrazi, S.A., Lian, Y.L.A., Ling, L.Y., Ibrahim, J. and Mahmud, R. (2012) Differentiating Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii Using Nested Polymerase Chain Reaction (PCR) in Rural Communities in Malaysia. Parasites & Vectors, 5, Article No. 187. https://doi.org/10.1186/1756-3305-5-187
[59]	Kim, H.S., Kim, D.M., Neupane, G.P., Lee, Y.M., Yang, N.W., Jang, S.J., et al. (2008) Comparison of Conventional, Nested, and Real-Time PCR Assays for Rapid and Accurate Detection of Vibrio vulnificus. Journal of Clinical Microbiology, 46, 2992-2998. https://doi.org/10.1128/JCM.00027-08
[60]	Carr, J., Williams, D.G. and Hayden, R.T. (2010) Molecular Detection of Multiple Respiratory Viruses. In: Grody, W.W., Kiechle, F.L., et al., Eds., Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory, Academic Press, Cambridge, 289-300. https://doi.org/10.1016/B978-0-12-369428-7.00024-0
[61]	Ali, I.K.M. and Roy, S. (2020) A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species that Infect Humans. Journal of Clinical Microbiology, 59, Article ID: e01986-20. https://doi.org/10.1128/JCM.01986-20
[62]	Singh, A., Banerjee, T., Khan, U. and Shukla, S.K. (2021) Epidemiology of Clinically relevant Entamoeba spp. (E. histolytica/dispar/moshkovskii/bangladeshi): A Cross Sectional Study from North India. PLoS Neglected Tropical Diseases, 15, Article ID: e0009762. https://doi.org/10.1371/journal.pntd.0009762
[63]	Hamzah, Z., Petmitr, S., Mungthin, M., Leelayoova, S. and Chavalitshewinkoon-Petmitr, P. (2006) Differential Detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii by a Single-Round PCR Assay. Journal of Clinical Microbiology, 44, 3196-3200. https://doi.org/10.1128/JCM.00778-06
[64]	Ferrer, A., Nguyen-Viet, H. and Zinsstag, J. (2012) Quantification of Diarrhea Risk Related to Wastewater Contact in Thailand. EcoHealth, 9, 49-59. https://doi.org/10.1007/s10393-012-0746-x
[65]	Berglund, B., Dienus, O., Sokolova, E., Berglind, E., Matussek, A., et al. (2017) Occurrence and Removal Efficiency of Parasitic Protozoa in Swedish Wastewater Treatment Plants. Science of the Total Environment, 598, 821-827. https://doi.org/10.1016/j.scitotenv.2017.04.015
[66]	Bahrami, F., Haghighi, A., Zamini, G. and Khademerfan, M. (2019) Differential detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii in fecal Samples Using Nested Multiplex PCR in West of Iran. Epidemiology and Infection, 147, Article No. e96. https://doi.org/10.1017/S0950268819000141
[67]	Mohammed, F.A., Taha, A.A. and Salama, M.A. (2017) Differentiation of Entamoeba histolytica from Entamoeba dispar by Nested Multiplex Polymerase Chain Reaction. Parasitologists United Journal, 10, 23-29. https://doi.org/10.21608/PUJ.2017.4732
[68]	Zebardast, N., Yeganeh, F., Gharavi, M.J., Abadi, A., Tabaei, S.J.S. and Haghighi, A. (2016) Simultaneous Detection and Differentiation of Entamoeba histolytica, E. dispar, E. moshkovskii, Giardia lamblia and Cryptosporidium spp. in Human Fecal Samples Using Multiplex PCR and qPCR-MCA. Acta Tropical, 162, 233-238. https://doi.org/10.1016/j.actatropica.2016.07.004
[69]	Khairnar, K., Parija, S.C. and Palaniappan, R. (2007) Diagnosis of Intestinal Amoebiasis by Using Nested Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Assay. Journal of Gastroenterology, 42, 631-640. https://doi.org/10.1007/s00535-007-2080-6
[70]	Obaid, H.M. (2016) Detection and Differentiation of Entamoeba histolytica and Entamoeba dispar by Enzyme Linked Immune-Sorbent Assay. Kirkuk University Journal for Scientific Studies, 11, 263-277. https://doi.org/10.32894/kujss.2016.124283
[71]	Shnawa, B.H. (2017) Molecular Diagnosis of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii: An Update Review. Annual Research & Review in Biology, 21, 1-12. https://doi.org/10.9734/ARRB/2017/37086
[72]	Akhtar, T., Khan, A.G., Ahmed, I., Nazil, R. and Haider, J. (2016) Prevalence of Amoebiasis in a Model Research Community and Its Confirmation Using Stool Antigen Elisa for Entamoeba histolytica. Pakistan Journal of Pharmaceutical Sciences, 29, 1587-1590.
[73]	Dhanalakshmi, S. and Parija, S.C. (2016) Seroprevalance of Entamoeba histolytica from a Tertiary Care Hospital, South India. Tropical Parasitology, 6, 78-81. https://doi.org/10.4103/2229-5070.175116
[74]	Santos, R.V., Fontes, G., Duarte, I.A., Santos, J.A. and Rocha, E.M.M. (2016) Identification of Entamoeba histolytica and Entamoeba dispar Infection in Maceio, Alagoas State, Northeast Brazil. The Journal of Infection in Developing Countries, 10, 1146-1150. https://doi.org/10.3855/jidc.6781
[75]	Chauret, C., Springthorpe, S. and Sattar, S. (1999) Fate of Cryptosporidium Oocysts, Giardia Cysts, and Microbial Indicators during Wastewater Treatment and Anaerobic Sludge Digestion. Canadian Journal of Microbiology, 45, 257-262. https://doi.org/10.1139/w99-001
[76]	Jiménez-Cisneros, B., Maya-Rendon, C. and Salgado-Velázquez, G. (2001) The Elimination of Helminth Ova, Fecal Coliforms, Salmonella and Protozoan Cysts by Various Physicochemical Processes in Wastewater and Sludge. Water Science and Technology, 43, 179-182. https://doi.org/10.2166/wst.2001.0733
[77]	Bitton, G. (2005) Wastewater Microbiology. 3rd Edition, John Wiley and Sons, Inc., Hoboken. https://doi.org/10.1002/0471717967
[78]	Leong, L. (1983) Removal and Inactivation of Viruses by Treatment Processes for Potable Water and Wastewater—A Review. Water Science and Technology, 15, 91-114. https://doi.org/10.2166/wst.1983.0043
[79]	US Army (1944) Efficiency of Standard Army Water Purification Equipment and of Diatomite Filter in Removing Cysts of Entamoeba histolytica from Water. The Engineer Board, Corps of Engineers, US Army, Fort Belvoir.
[80]	Schaefer, F.W., Marshall, M.M. and Clancy, J.L. (2004) Inactivation and Removal of Enteric Protozoa in Water. In: Sterling, C.R. and Adam, R.D., Eds., The Pathogenic Enteric Protozoa: Giardia, Entamoeba, Crysptosporidium and Cyclospora. World Class Parasites, Vol. 8, Springer, Boston, 117-127. https://doi.org/10.1007/1-4020-7878-1_9
[81]	Jimenez, B., Chavez, A. and Hernandez, C. (1999) Alternative Treatment for Wastewater Destined for Agricultural Use. Water Science and Technology, 40, 355-362. https://doi.org/10.2166/wst.1999.0610
[82]	Shukla, S., Arora, V., Jadaun, A., Kumar, J., Singh, N. and Jain, V.K. (2015) Magnetic Removal of Entamoeba Cysts from Water Using Chitosan Oligosaccharide-Coated Iron Oxide Nanoparticles. International Journal of Nanomedicine, 10, 4901-4917. https://doi.org/10.2147/IJN.S77675
[83]	Mtapuri-Zinyowera, S., Midzi, N., Muchaneta-Kubara, C.E., Simbini, T. and Mduluza, T. (2009) Impact of Solar Radiation in Disinfecting Drinking Water Contaminated with Giardia duodenalis and Entamoeba histolytica /dispar at a Point-of-Use Water Treatment. Journal of Applied Microbiology, 106, 847-852. https://doi.org/10.1111/j.1365-2672.2008.04054.x
[84]	Mohamed, A.H., Galal, M., Osman, G., Y. and Aboamer, M. (2014) Influence of Solar Energy on Encyste Parasitic Protozoa Giardia and Entamoeba spp in Water-Treatment Plants, Egypt. Journal of Materials Science and Engineering B4, 10, 284-292. https://doi.org/10.17265/2161-6221/2014.10.003
[85]	Maya, C., Beltran, N., Jimenez, B. and Bonilla, P. (2003) Evaluation of the UV Disinfection Process in Bacteria and Amphizoic Amoebae Inactivation. Water Science and Technology: Water Supply, 3, 285-291. https://doi.org/10.2166/ws.2003.0074
[86]	Ryu, H., Gerrity, D., Crittenden, J.C. and Abbaszadegan, M. (2008) Photocatalytic Inactivation of Cryptosporidium parvum with TiO2 and Low-Pressure Ultraviolet Irradiation. Water Research, 42, 1523-1530. https://doi.org/10.1016/j.watres.2007.10.037
[87]	Sokmen, M., Degerli, S. and Aslan, A. (2008) Photocatalytic Disinfection of Giardia intestinalis and Acanthamoeba castellani cysts in Water. Experimental Parasitology, 119, 44-48. https://doi.org/10.1016/j.exppara.2007.12.014
[88]	Mendez-Hermida, F., Ares-Mazas, E., McGuigan, K.G., Boyle, M., Sichel, C. and Fernandez-Ibanez, P. (2007) Disinfection of Drinking Water Contaminated with Cryptosporidium parvum Oocysts under Natural Sunlight and Using the Photocatalyst TiO2. Journal of Photochemistry and Photobiology B: Biology, 88, 105-111. https://doi.org/10.1016/j.jphotobiol.2007.05.004
[89]	National Research Council (1986) Drinking Water and Health. Vol. 2, National Academy Press, Washington DC.
[90]	Centers for Diseases Control and Prevention (2012) Effect of Chlorination on Inactivating Selected Pathogen. https://cdc.gov/safewater/effectiveness-on-pathogens.html
[91]	Rubin, A.J., Engel, J.P. and Sproul, O.J. (1983) Disinfection of Amoebic Cysts in Water with Free Chlorine. Water Pollution Control Federation, 55, 1174-1182.
[92]	Stringer, R.P., Cramer, W.N. and Kruse, C.W. (1975) Comparison of Bromine, Chlorine and Iodine as Disinfectants for Amoebic Cysts. In: Johnson, J.D., Ed., Disinfection: Water and Wastewater, Ann Arbor Science, Ann Arbor, 193-209.
[93]	Brady, F.J., Jones, M.F. and Newton, W.L. (1943) Effect of Chlorination of Water on Viability of Cysts of Endamoeba histolytica. War Medicine, 3, 409-419.
[94]	WHO (World Health Organization) (2018) Alternative Drinking-Water Disinfectant, Part 1. World Health Organization, Geneva.
[95]	KingNod (2021) The Disinfectant of Bromine. https://ikingnod.com/the-disinfectant-of-Bromine
[96]	Chang, S.L. (1966) Iodination of Water (Yodacion del agua). Pan American Sanitary Bureau, 6, 317-324.
[97]	WHO (World Health Organization) (2016) Iodine as a Drinking Water Disinfectant. Vol. 2, World Health Organization, Geneva.
[98]	Hoseinzadeh, E., Rostamian, A., Razaghi, M. and Wei, C. (2021) Waterborne Transmission of Protozoan Parasites: A Review of Water Resources in Iran—An Update 2020. Desalination and Water Treatment, 213, 91-105. https://doi.org/10.5004/dwt.2021.26678
[99]	Newton, W.L. and Jones, M.F. (1949) Effect of Ozone in Water on Cysts of Entamoeba histolytica. The American Journal of Tropical Medicine and Hygiene, 29, 669-681. https://doi.org/10.4269/ajtmh.1949.s1-29.669
[100]	Awuah, E. (2006) Pathogen Removal Mechanisms in Macrophyte and Algal Waste Stabilization Ponds. Routledge, the Netherlands.
[101]	Rawat, K.P. and Sarma, K.S.S. (2013) Enhanced Biodegradation of Wastewater with Electron Beam Pretreatment. Applied Radiation and Isotopes, 74, 6-8. https://doi.org/10.1016/j.apradiso.2012.12.013