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] .

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 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] .

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,

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).

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,

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] .

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.

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.

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

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.