Entamoeba histolytica in Different Water Sources of Niğde Province of Turkey

Purpose This study was carried out to determine the presence of Entamoeba histolytica in water sources of Niğde province in Turkey, between June and November 2021. Methods A total of 90 water samples were taken from 15 different water sources (drinking water, well water, spring water, wastewater and dam water) every month and the presence of E. histolytica antigens in the samples was examined by ELISA. Results The positivity for E. histolytica was determined in 7 (7.7%) of 90 samples. While no antigens were found in any of the samples in June and September, E. histolytica was positive for three samples (20%) in July, one sample (6.6%) in August and October and two samples in November (13.3%). One of 24 dam samples (4.1%), 1 of 12 wastewater samples (8.3%), 1 of 12 well samples (8.3%), and 4 of 24 fountain samples (16.6%) that examined by ELISA were found positive. On the other hand, none of the examined 18 spring samples were positive. In addition, 4 (8.8%) of 45 samples that examined in summer and 3 (6.6%) of 45 samples that examined in autumn were detected positive by using ELISA. Entamoeba histolytica positivity in samples was statistically insignificant in terms of months, water resources and seasons (P > 0.05). Conclusion As a result, the presence of E. histolytica, which is an important public health problem in water sources, was determined for the first time in Niğde province of Türkiye with this study.


Introduction
Amebiasis is one of the protozoan diseases in the intestines caused by E. histolytica.Entamoeba histolytica is more prevalent in developing countries and is generally transmitted to humans through cysts found in feces or contaminated food and water [1].It is estimated that approximately 10% of the world's population is infected with this parasite, with distribution in Turkey ranging from 0 to 17% [2,3].While E. histolytica primarily infects humans, it can also parasitize various monkey species, dogs, cats, pigs, cattle, and rats.However, it is noted that the parasite does not form cysts

Study Area
Niğde is located at an altitude of 1,299 m in the Central Anatolia Region of Türkiye.According to the 2020 statistics, the population of Niğde city center is 230.776people.Niğde experiences the typical continental climate of Central Anatolia, with hot and dry summers and cold and snowy winters.Rainfall is generally in the form of snow in winter and rain in the spring.The average temperatures indicate that July is the hottest month, while January is the coldest.Showers are observed in the form of heavy rain in April, May, and the early days of June.The average precipitation in the city is 0.9 mm.April has the highest rainfall with 78.5 mm, while July has the lowest with 0.2 mm.

Field Study
Water samples were collected from 15 different water sources every month, between June 2021 and November 2021 in the Niğde province.The samples were collected in 5 L clean plastic containers.The collected water samples included 4 from dam waters used for agricultural irrigation, 2 each from wastewater and well water, 3 from spring water, and 4 from fountains used for drinking water purposes.

Laboratory Study
The sample number was chosen considering these many samples were proven to represent the population adequately in previous studies [8,9].A total of 90 water samples were stored at 4 °C until they were processed in the laboratory.A vacuum pump filtration device with a 0.45 μm cellulose acetate membrane filter (Ø, 45 mm) was used for filtering the water samples.Subsequently, the filter was transferred to a clean tube, and 20 ml of the same water sample was added.After vortexing to remove particulate matter, the sample was centrifuged at 2500 rpm for 10 min to discard the upper portion, and the 1.5 ml sediment at the bottom was transferred to an eppendorf tube and stored in a deep freezer at -20℃ until further analysis with the ELISA.

ELISA (Enzyme Linked Immunosorbent Assay)
A commercial ELISA kit (E.histolytica II™, TECHLAB 2001 Kraft Drive Blacksburg, VA 24060 − 6358 USA) was used to investigate the specific adhesin antigen of E. histolytica in samples collected from different water sources.The ELISA was performed following the procedure provided by the manufacturer.
ELISA results were read at 450 nm wavelength using an ELISA microplate reader, MR-96 A, and the obtained values were calculated using the kit procedure.

Statistical Analysis
In the conducted study, the chi-square test was used for the statistical evaluation of positivity values concerning the months of water sample collection, water sources, and seasons.Therefore, the SPSS (Statistical Programme for Social Science) for Windows 24.0 Statistical Package Program was utilized.

Results
Out of the total 90 water samples collected from 15 different water sources in the Niğde province, the presence of E. histolytica was detected in seven samples (7.7%) through the ELISA.
In Table 1, the presence of E. histolytica is given according to the months.Accordingly, for water samples taken from 15 different sources, in June and September, none of them showed E. histolytica presence.In July, three samples (Uluagac, Yesilburc, Okcu) showed positivity at a rate of 20%.In August (Kemerhisar) and October (Alaaddin), one sample each showed positivity at a rate of 6.6%.In November, two samples (Alaaddin, Kizilca Creek) showed positivity at a rate of 13.3%.Entamoeba histolytica positivity of water samples by months was found to be statistically insignificant (p > 0.05).
In Table 2, E. histolytica presence is presented according to the water sources from which the samples were collected.Accordingly, out of the 24 dam water samples examined with the ELISA test, 1 (4.1%) was found to be positive.Among the 12 wastewater samples, 1 (8.3%) was positive, as well as 1 (8.3%) out of the 12 well water samples.Additionally, 4 (16.6%)out of the 24 fountain water samples were found to be positive.In contrast, none of the 18 spring water samples examined showed the presence of E. histolytica.When the collected water samples were examined in terms of E. 1 3 histolytica positivity rates based on water sources, it was determined to be statistically insignificant (p > 0.05).
In Table 3, E. histolytica presence determined according to seasons for the collected water samples is provided.Accordingly, out of the 45 water samples examined with the ELISA test during the summer months, 4 (8.8%) were positive, and out of the 45 samples examined in the fall months, 3 (6.6%)were positive.The presence of E. histolytica was detected in water samples during the summer and fall months, and the positivity determined in terms of seasons was found to be statistically insignificant (p > 0.05).

Discussion and Conclusion
Amebiasis caused by E. histolytica is widespread in Turkey and worlwide.Amoebic dysentery is frequently occured in developing and underdeveloped countries.Entamoeba histolytica exists in our environment as a cyst form and is transmitted to humans orally by inadequately sterilized vegetables, fruits, and drinking water [3,10].
In Turkey, the presence of protozoan parasites in different water sources revealed protozoa such as G. intestinalis, C. parvum and T. gondii that have been investigated more than E. histolytica [24][25][26][27][28][29][30][31].In a study conducted in Ankara, samples taken from various water sources were examined for standard microscopic, IFA, ELISA, and PCR techniques.Two of the examined well water samples were found to have G. intestinalis, two of the river water samples had E. histolytica, and one had C. parvum, no parasites were detected in municipal waters and dam water [8].In this study, however, it was found that 7 out of a total of 90 water samples taken from 15 different water sources in Niğde province were positive for E. histolytica by ELISA test, and the prevalence of E. histolytica was determined to be 7.7%.Of the positive samples, 4 were neighbourhood fountains (Yesilburc, Okcu, and Alaaddin), 1 was a dam (Uluagac), 1 was well water (Kemerhisar), and 1 was wastewater (Kizilca Creek).It was observed that the results obtained from various water sources for E. histolytica in this study conducted in the Niğde province differed from the results in Ankara.The difference may be due to the contamination levels of the water samples, the disinfection methods applied, and the detection methods used.
Amebiasis caused by E. histolytica is widespread worldwide, it is more common in tropical and subtropical regions.In Niğde region, the presence of E. histolytica in humans was found microscopically in fecal samples of university students at a rate of 2.12% [32], and in primary school students at a rate of 13.34% [33].This situation suggests that people in the Niğde province are infected with this parasite (2.12-13.34%),and when there is a problem in the sewage systems, water sources may become contaminated fecally.Thus, waters containing E. histolytica may pose a risk to public health.
In conclusion, the presence of E. histolytica in different water sources (drinking water, well water, spring water, wastewater, and dam water) in the Niğde province is reported for the first time in this study.The presence of E. histolytica in 7.7% of water sources was determined by ELISA.ELISA is among the most popular methods used in diagnostic laboratories throughout the world.When the test protocol is applied correctly, ELISA kits have very high sensitivity and specificity (> 95%).The E. histolyticaspecific ELISA offers the significant advantage of the rapid differentiation of E. histolytica from E. dispar [34,35].In addition, further investigations, including molecular techniques are required in order to determine the importance of this parasite as a cause of clinical disease in human in Niğde province.However, it is less expensive and relatively easier to detect the protozoan species with ELISA compared to PCR analysis.This study emphasize the importance of an adequately and appropriately disinfection treatment of drinking water, well water, wastewater, and dam waters in terms of public health in the Niğde province.It is crucial to increase public awareness of the impact of E. histolytica on public health in the Niğde province to contribute to the prevention of waterborne parasitic epidemic diseases and ensure people's access to safe and healthy water.

Table 1
Entamoeba histolytica presence in water sources with regard

Table 2
Entamoeba histolytica presence according to the sampled

Table 3
Entamoeba histolytica presence according to seasons