Prevalence and associated risk factors of Entamoeba histolytica infection among school children from three primary schools in Arsi Town, West Zone, Ethiopia

Entamoeba histolytica is one of the major pathogenic intestinal parasites and is amongst the leading causes of diarrheal illness in children. Around 500 million people are infected worldwide, while 75,000 die of the disease annually. E. histolytica is associated with moderate to severe diarrhoea and increased mortality among children in African countries and negatively affects child growth and development. Malnutrition is also an important contributor to the prevalence of E. histolytica in Ethiopia. It is mostly associated with poor hygiene, poverty, illiteracy, lack of access to potable water, and a hot, humid tropical climate. Thus, the present investigation was aimed to assess the prevalence rate and associated factors of E. histolytica among schoolchildren in Arsi Town, West Zone, Oromia Regional State, Ethiopia. A cross-sectional study was conducted from February 1st to May 1st, 2020. A total number of 334 students were selected from three governmental elementary schools in the west zone using a systematic sampling method. A structured questionnaire was adopted to identify environmental, socio-demographic, and behavioral factors. Microscopically positive samples for E. histolytica cysts were further characterised using an E. histolytica II antigen detection kit. The statistical analysis of the data was done using SPSS software. A univariate and multivariate logistic regression analysis was done. P value < 0.05 was considered statistically significant. The overall prevalence of E. histolytica was 19.8%. Hand washing habits before eating [AOR = 0.32; 95% CI (0.12, 0.84)], hand washing habits after defecation [AOR = 0.396; 95% CI (0.165, 0.946)], and ameba awareness [AOR = 0.35; 95% CI (0.142, 0.889)] were factors associated with parasite prevalence. The findings of this study could assist the government in targeting infected areas, improving sanitation to prevent E. histolytica transmission, and implementing effective control measures in these rural communities, particularly among youngsters, who represent the nation's future.


Introduction
Amebiasis is a disease caused by the intestinal parasite Entamoeba histolytica, has an estimated worldwide prevalence of 500 million cases of symptomatic disease, and 40.000-110.000 deaths annually .It is the 3rd most common parasitic cause of death worldwide (Schmidt and Roberts2000;Stanley and Jr 2003). Disease caused by E. histolytica characterized by self-limited diarrhea but can evolve to long-term complications and malnutrition is one of them. The lifecycle of Entamoeba histolytica involves two distinct morphogenetic stages: the amoeboid and proliferative trophozoite and the infectious cyst form (Chou and Austin 2021). Infections in humans begin with the ingestion of viable cysts in food or water that has been contaminated by feces. It colonizes the intestinal tract, leading mainly to tissue destruction and secretory bloody diarrhoea (Mendoza Cavazos & Knoll 2020).
Entamoeba histolytica (Amoebiasis) occurs worldwide; however, the level of prevalence and presentation of symptoms of infection varies geographically. E. histolytica has been covered worldwide, infecting approximately 50 million people annually, causing close to 100,000 deaths per year (WHO 1997). Based on a retrospective examination of the known literatures, a global estimate of amebiasis was made in 1986, and it was estimated that around 500 million people were infected with E. histolytica worldwide (Walsh 1986). The infection is more prevalent in the tropics and sub-tropics (area of poor sanitation and nutrition) than in colder climates. Morbidity and mortality are present in Africa, Asia, Central and South America (Petri and Singh 2006).
Entamoeba. histolytica differs among countries, socioeconomical and aseptic conditions, and populations. It is highly pandemic throughout poor and socio-economically deprived communities in the tropics and subtropics. Environmental, socio-economic, demographic, and hygienerelated behavior is known to impact the transmission and distribution of intestinal parasitic infections. Many studies identified that place of residence, age, eating raw vegetables, lack of toilet facility and drinking water quality as important risk factors (Adeyeba and Akinlabi 2002).
As per the UNISEF survey, diarrhea (bloody diarrhea due to amoebiasis infection) is the leading causes of underfive mortality in Ethiopia, accounting for 23 percent, more than 70,000 under age ten children death a year. In Ethiopia, 60-80 percent of infectious diseases are due to limited access to potable water, inadequate sanitation and hygiene services. In addition, an estimated 50 percent of the consequences of under nutrition are caused by socioeconomic factors like poor hygiene and lack of access to water supply and sanitation. There are strong relationship between sanitation and stunting, whereas, open defecation can lead to fecal-oral diseases such as diarrhea which is a one of major cause of malnutrition [https://www. unicef.org/ethiopia/water-sanitation-and-hygiene-wash; Beyene et al. 2015). According to survey report of 97 communities of Ethiopia, the overall prevalence of Entameba histolytica infections recorded was 15.0% in schoolchildren (Erko et al. 1995). In Ethiopia, intestinal parasitic infections (IPIs) including Entamoeba infections are more prevalent in rural areas due to poverty, illiteracy, poor hygiene, lack of access to potable water and hot and humid tropical climate (Mengistu and Berhanu 2004).
Although several studies have been conducted on the distribution and prevalence of amebiasis in Ethiopia, still several localities including study area there is lack of data regarding amoebiasis-related morbidity and mortality. We analyzed death certificate data from the last ten years to assess the prevalence of amoebiasis-related deaths. A total of 248 amoebiasis-related deaths were identified and decided to conduct a study to assess the prevalence, characteristic nature, and risk factors of E. histolytica infections among the school children.

Methodology Description of study area
The proposed study was conducted in Arsi Town, West Zone, Oromia Regional State, Ethiopia. The place is located has a longitude and latitude of 7°21 0 N 38°42 0 E with an elevation of 2043 m above sea level. In a town, there are six government elementary schools. The three schools, Ashale, Hadansho, and Mako elementary school were included in this study. As per the school record, Ashale 4279 (Male = 2054 and Female = 2225) students, Hadansho has 2717 (Male = 1360 and Female = 1357) students and Mako elementary school has 3776 (Male = 1808 and Female = 1968) students. The age of the students ranged from 7-20 years.

Research design and study population
A school based cross-sectional study was conducted to establish the prevalence of E. histolytica infection and associated risk factors among three randomly selected primary school children from Arsi town by simple random and stratified sampling techniques. The students were first stratified according to their educational level (Grade 1 to Grade 8) in selected school and allocation of students was done in proportional to the number of students in each school and grade level. Finally, the students were selected using systematic random sampling by using class roster as the sampling frame.
Based on the calculation the total sample size for the study was 334. The actual number of students who participated in the study was selected using a simple random sampling technique to incorporate 334 students.

Stool samples and data collection
After giving proper instruction on how to collect the stool sample, each school children were supplied the clean, dry collection cups with unique identification numbers, with applicator sticks and instructed to bring at least 10 g of proper fresh stool sample in small bottles. Date of sampling, number of the participant, grade, section, school name, age, and sex were recorded. Each sample was divided into 2 parts. First part immediately was transported to the laboratory and examined within 30 min of collection of the sample to avoid disintegration of trophozoites. Fecal samples (formal-ether concentration) were examined for the presence of ova, cysts, and larvae. Second part of unfixed stool sample was stored at -20°C for performing Entamoeba histolytica antigen detection test (Parija et al. 2014).
Data on demographic and determinant was collected by trained laboratory assistant by using a structured questionnaire. The questionnaire was prepared originally in English and then translated into the local language of Oromo and Amharic.

Laboratory analysis procedures
The stool samples were processed by direct wet smear and formalin ethyl acetate sedimentation technique followed by iodine staining and examined via microscopy for the presence of E. histolytica (Fig. 1). Microscopically positive samples for E. histolytica cysts were further characterized using antigen detection kit. Diagnosis was based on the identification of Entamoeba histolytica worm eggs or larvae, protozoan trophozoites, and cysts.

Entamoeba histolytica antigen detection
The antigen detection E. histolytica II test (TechLab, Inc., Blacksburg, VA) was performed on unfixed fresh stool samples containing E. histolytica cysts according to the manufacturer's instructions. This was done to detect the non-pathogenic form of Entamoeba (E. dispar) (Haque et al. 2000). The kits were provided by USAID under Health Workforce Improvement Program in Ethiopia.

Data analysis
Data was entered and analyzed using SPSS software version 20.0. Descriptive statistics was done to calculate frequencies and percentages. The associations of potential risk factors and presence of E. histolytica infection were performed by binary logistic regression analysis model (Hosmer and Lemeshow 2000). Multivariate logistic regression analysis was used to measure the strength of association of socio-demographic factors and E. histolytica infection. Ninety five percent confidence interval (CI) was calculated for the odds ratio value. The results of the association were considered as significant when the p value was below 0.05.

Socio-demographic characteristics of the study subjects
Three hundred thirty four, school children were recruited and included in the analysis, with a 100% response rate. There were 165 (49.4%) males and 169 (50.6%) females. The major age ranges of the participants were from five to seventeen years. Most of the study participants, 170 (50.9%) were 1 to 4 grade level. Regarding the educational status of the father's, above diploma was 94 (28.1%). 110 (32.9%) mother's educational level was limited up to write and read only. 39.5 percent of fathers were merchant whereas 42.9 percent of mothers were housewife by occupation (Table 1).

Frequency distribution of behavioral and environmental related factors
A large number, 312 (93.4%) of the students had tap water of drinking water source. Regarding drinking water storage, 62.3% of the households had jerican and 6% of the Fig. 1 Morphology of the diagnostic stage of Entameba histolytica, cyst household keeps the water uncovered. 76.4% did not treat the drinking water by chlorine or boiling. 100% had the toilet facility at home constructed in and around the compound. Of households who had toilets, 59.3% were lacking water access in toilet. 66.5% of household had no sewage disposal facility, 31.7% of the student had no habit of washing hands before eating whereas, 21.3% had the habit of washing hands after detraction with soap. 91.0% of the study participants were aware about the importance of nail trimming. Only 21.0% had awareness about amoebiasis ( Table 2).

Prevalence of E. histolytica
Microscopy and E. histolytica II antigen detection kit were used to test single stool specimens from 334 asymptomatic school children aged 5 to 17 years. Antigen detection test was positive in 66 (19.76%) of stool specimens (Table 3). Among the study subjects, 165 were males and 169 were females. Higher prevalence of E. histolytica was found in males compared with female students. Regarding, the E. histolytica infection in specific age group, children with age group 5-9 was more infected than other age groups of study subjects but this is not significant difference due to unequal sample size. Out of 334 students, 84, 117, and 133 were belongs to Ashale, Hadansho and Mako elementary school respectively. The highest prevalence was observed in Ashale primary school children ( Fig. 1; Table 3).

Association of risk factors with E. histolytica infection among school children
In this study significant relationships were observed between E. histolytica infection and dependent variable and some of independent variables. The result showed that mother's occupation in the form of farmer, hand wash before eating, hand wash after defecation and awareness about amoebiasis showed significant association ( P \ 0.05) with prevalence of E. histolytica in school children (Tables 4 and 5).

Discussions
Amoebiasis is the second leading cause of death from parasitic disease globally. Entamoeba histolytica the causative protozoan parasite is a potent pathogen and very infrequent cause of childhood diarrhea or dysentery in developing countries (Black and Lanata 2002 (Ngui et al. 2011) and up to 30% in Iran (Nazarian 1973;Sheiban and Rezaian 1981 (Ngui et al. 2012). This study extends the observation that, infection of E. histolytica was more prevalent in male student 34 (20.6%) than female 32 (18.9%), however, it was not statistically significant. There was no any significant association between sex and prevalence of the parasite. These findings are supported by results of Zeb et al. (2018) and Amaechi et al. (2017). This difference might be due to male have more outdoor works in villages and expose to the more unhygienic environment (Abossie and Seid 2014). This result was inconsistent with the study conducted in Benishangul Gumuz which observed that the prevalence rate is higher in female students (39.36%) as compared to male students (31.88%) (Gebremichael 2016).
Results also show that E. histolytica found more prevalent in 5-9 age groups (20.6%) than [ 15 (16.7%) age group. But, there was no statically significant association between prevalence and age. This finding is further confirmed with the study done in Pakistan (Zeb et al 2018), and in Nigeria (Amaechi et al. 2017).
Many studies reported that there was a web of risk factors associated with the high prevalence of protozoa and other parasitic infections which includes habit of hand washing before eating, hand washing after defection, age, inadequate sanitation, untreated water supply, low level of Most amoebic studies that identified Entamoeba histolytica infection in Ethiopia were performed without the distinction of the separate species. This is possibly due to the lack of laboratory facilities and well trained technical staff. This investigation represents the first time research to use antigen detection kit. In the present study, the prevalence rate of E. histolytica was different by microscopy and antigen detection kit. Those samples were positive by microscopy again tested by antigen detection test, each was found negative. It is well known fact that diagnosis of E. histolytica by microscopic examination is based on protozoan morphology and unable to differentiate among protozoa with similar morphological features. It is also possible that the samples which were positive by microscopy but not antigen detection test may belong to other common amoebas. It proves that diagnostic sensitivity and specificity of microscopic examination to detect E. histolytica in stool is low (Haque et al. 1995;WHO 1997;Tanyuksel and Petri 2003; but one study observed that TechLab enzyme-linked immunosorbent assay (ELISA) was less sensitive than microscopy (Fotedar et al. 2007).Therefore, a more sensitive method such as Real-Time PCR with specific primers for all amoebas species should be considered in future study.

Conclusions and recommendations
Amoebiasis infections is major public health concerns and highly prevalent among the poor and socioeconomically deprived rural and remote communities of Africa including Ethiopia. According to several studies it is associated with poverty, poor environmental sanitation and lack of clean water supply; it is highly needed that these factors should be addressed effectively. Development of socioeconomic status, sanitation, health education to promote awareness about health and hygiene together with mass deworming under national health development programme are better strategies to control these infections. The present study could help the government to hit the infected areas; improve sanitation to reduce the level of transmission of E. histolytica with effective control measures in, these rural community, especially children who are the future of nation.
Funding No specific fund was received for the proposed work.
Data availability Not applicable. However, raw data can be obtained from the corresponding author upon request.

Declarations
Conflict of interest The Authors declare that they have no conflict of interest.
Ethical approval The study protocol was approved by Dilla University Ethical Committee (DUEC Ref Du/241/21). Oral consent had been taken from school directors. Informed consent was obtained from all students recruited in the study. Written informed consent was obtained from the parents. The questionnaires concerning the prevalence of study were recorded during sample collection. Apart from these, students were asked to fill the questionnaire and assist during sample collection. The information obtained during the study period was kept confidential. Paper data were secured in a locked cabinet confidentially and computer based data were protected with passwords. Except the research team members, no one has permission to access patient data.