Prevalence of Entamoeba histolytica and Giardia lamblia among schoolchildren in Um-Asher Area, Sudan

Objective: Infections with Entamoeba histolytica and Giardia lamblia are widely prevalent and responsible for serious public health issues among school-aged children in developing countries. This study was conducted to determine the prevalence of these parasites among children from two primary schools in Um-Asher area, Khartoum, Sudan. A total 170 fresh stool specimens were collected from November 2017 to June 2018. The samples were examined by microscopy for the presence of the two parasites. Results: The overall prevalence of both parasites among the 170 children was 20%, with 13 children infected with E. histolytica (7.6%) and 23 with G. lamblia (13.5%). Most of the positive cases were single infections. Only two children (1.2%) had mixed infections. This indicates that these gastrointestinal parasites remain a challenging public health concern wherever sanitation and health measures are limited.


Introduction
Intestinal parasites are a serious medical problem that signi cantly affects public health and results in considerable morbidity and mortality in developing countries (1), including Sudan (2,3), where many conditions, including climatic, ecological, socioeconomic and hygienic, favour their transmission. In addition, infection with these pathogenic parasites can be associated with gastrointestinal diseases and malnutrition, particularly among young children (4). Several pathogenic intestinal parasites may be responsible for the above health problems, two parasites, Entamoeba histolytica (E. histolytica) and Giardia lamblia (G. lamblia) being the commonest and have been reported and associated with signi cant illnesses in developing countries. E. histolytica, the etiologic agent of amoebiasis, is an important parasite of the human gut. Among parasitic diseases, amoebiasis is considered the third most common cause of death after malaria and schistosomiasis (5). The parasite causes amoebic dysentery, amoebic colitis and amoebic liver abscess and leads to almost 100 thousand deaths annually (6). G. lamblia is another common cause of gastrointestinal diseases in humans throughout the world, and approximately 5-10% of the global population is at risk of giardiasis, which is considered one of the main nonviral causes of diarrhoeal diseases in humans (7).
In Sudan, the two parasites are widely distributed and pose signi cant challenges to health authorities due to insu cient health education, poor sanitation and improper disposal of human excreta, which lead to the contamination of food and drinking water, and poverty (1,8). In this regard, Sudanese children are at risk of infection (9); the infection rate of Entamoeba spp. is predicted to increase. Meanwhile, schoolchildren are the primary victims of Giardia spp. infection in both developed and developing countries. Accordingly, prevalence surveys of both parasites are a prerequisite for developing effective control strategies. However, only few studies have been conducted in different parts of Sudan and mostly among schoolchildren. Therefore, this study was conducted to determine the prevalence of E. histolytica and G. lamblia and the associated risk factors among schoolchildren from Um-Asher area, South Khartoum, Sudan.

Methodology
Study area, period and study subjects A cross-sectional study conducted in Um-Asher (Al Kalakla) area, from November 2017 until June 2018.
Um-Asher is located at the latitude of 15°28′04 N and longitude of 32°29′08 E, a height of 384 m and 16.3 km south of Khartoum, Sudan. Convenience sampling method was used to enrol children aged between 6 and 17 years who were attending the selected primary schools. Any children who were taking medication for amoebiasis or giardiasis within the three weeks of data collection were excluded.
Accordingly, 170 primary schoolchildren (75 males and 95 were females) were selected and enrolled.

Sample collection and examination
Approximately 5-7 g of fresh stool specimens were collected in sterile, carefully labelled plastic containers. The stool sample was added with 10% formaldehyde as preservative. The stool specimens were individually inspected for the presence of mucous or blood and consistency. The detection of E. histolytica and G. lamblia infections was carried out using direct faecal smears in normal saline. All patient details including socio-demographic characteristics and associated risk factors were obtained via previously developed standardised questionnaire (10).
Statistical analysis SPSS 24.0 was used to conduct the descriptive and logistic regression analyses. The descriptive data were presented as frequencies and percentages. Simple and multiple logistic regression analyses were performed to identify variables associated with the positive outcome of E. histolytica and G. lamblia. In the simple logistic regression analysis, the variables with a p-value < 0.25 were considered important and therefore included in the multiple logistic regression analysis. The nal model was assessed for tness by using the receiver operating characteristics (ROC) curve, the Hosmer and Lemeshow test, the classi cation table, interaction between the study variables and multicollinearity.

Results
Socio-demographic characteristics of study participants A total of 170 students from two primary schools were involved. The majority of the participants (68.2%) were between the age of 10 and 13 years. Amongst the study participants, 33.5% (n = 57) were residing less than 1 km from a water source, and 66.5% (n = 113) were residing more than 1 km away from a water source. The main source of water was tap water 94.7% (n = 161). The majority of the participants (61.8%, n = 105) had no sanitary latrine. The summary of all the participant's characteristics is presented in Table 1. Prevalence of E. histolytica infection and its associated risk factors Microscopic analysis showed that only 13 (7.6%) were found positive for E. histolytica. In the simple logistic regression analysis, three variables, namely: gender, latrine type, and breakfast had an unadjusted crude odds ratio (COR) with p-values less than 0.25 (Table 2). Male were 3.1 times more likely to test positive for E. histolytica than female children (COR = 3.10, p-value = 0.069). The subjects who ate breakfast in the school were 7.8 times more likely to test positive than those who took breakfast at home (COR = 7.83, p-value = 0.051). Furthermore, subjects with no sanitary latrine were 64% less likely to test positive than those with sanitary latrine (COR = 0.36, p-value = 0.082). The forward LR and backward LR selection method was used in the multiple logistic regression analysis. All the three variables (gender, type of latrine and breakfast) were selected and run using the enter method. Male were 3.5 times more likely to test positive than female, with adjusted odds ratio (AOR) = 3.50 and p-value = 0.052. Subjects who took their breakfast in school were 9.5 times more likely to test positive than those who ate breakfast at home (AOR = 9.50, p-value = 0.035). Those with no sanitary latrine were 71% less likely to test positive than those with sanitary latrine (AOR = 0.29, p-value = 0.043).

Prevalence of G. lamblia infection and its associated risk factors
A total of 23 children (13.5%) were found to be infected with G. lamblia. In simple logistic regression analysis, three variables, namely: age, bathing habit, and frequency of trimming had an unadjusted COR with p-values less than 0.25, (Table 3). Children aged more than 10 years were 52% less likely to be diagnosed positive for G. lamblia than those aged 6-9 years (COR = 0.48, p-value = 0.130). For bathing habit, children who took their baths in the river were 13.1 times more likely to test positive than those who took their baths at home (COR = 13.10, p-value = 0.039). Children who trimmed their nails more than once in a week were 74% less likely to test positive than those who trimmed their nails once in a week (COR = 0.26, p-value = 0.019). In the multiple logistic regression analysis, two variables were selected and run using the enter method (age and frequency of trimming). Children aged more than 10 years were 67% less likely to be diagnosed positive for G. lamblia than those aged 6-9 years (AOR = 0.33, p-value = 0.035). The children who trimmed theirs nails more than once in a week were 80% less likely to be diagnosed positive for G. lamblia than those who trimmed their nails once in a week (AOR = 0.20, p-value = 0.008).
Children who trimmed their nails every two weeks were 91% less likely to be diagnosed positive than those who trimmed their nails every week (AOR = 0.09, p-value = 0.027). Children who trimmed their nails every three weeks were 79% less likely to test positive than those who trimmed their nails every week (AOR = 0.21, p-value = 0.158). Children who trimmed their nails every month were 57% less likely to test positive than those who trimmed their nails every week (AOR = 0.43, p-value = 0.315) (data not shown).
There was no signi cant interaction between the age and frequency of nail trimming (p-value = 0.376). No multicollinearity for all the variables (VIF < 10) was observed. The assumption for the Hosmer and Lemeshow test was satis ed (p-value = 0.663), indicating adequate model tness with the two variables.
The area under the ROC curve (AUC) was 68.5%, indicating the su cient discriminant ability of the nal model with age and frequency of nail trimming.

Discussion
Epidemiological information that con rms the spread of intestinal parasitic infections is a prerequisite in the design and implementation of appropriate prevention and control strategies. This study was conducted to determine the prevalence of two gastrointestinal parasites, i.e. E. histolytica and G. lamblia, and the associated risk factors among schoolchildren in Um-Asher area, South Khartoum, Sudan.
The overall prevalence of the two parasites among the 170 children was 20% (34 of 170), with 13 children infected with E. histolytica (7.6%) and 23 with G. lamblia (13.5%). Mixed infections with G. lamblia and E. histolytica were detected in 1.2% (2 of 170) of the children. G. lamblia was remarkably more frequent among the children than E. histolytica. This nding is consistent with the results of previous studies conducted in different parts of Sudan and globally. The predominance of G. lamblia over E. histolytica was also identi ed in a study conducted by Suliman et al. (11), Siddig et al. (12) and Gabbad and Elawad (13). Similar infection patterns were reported from other countries, such as Ethiopia (14), Nepal (15) and Iran (16).
The proportion of children infected with E. histolytica was higher than those infected with G. lamblia, similar to the report of a study conducted in the same geographical area (17) and in Côte d'Ivoire, Iraqi and Ethiopia (5,18,19). The discrepancy in prevalence and predominance of the two parasites among studies conducted inside or outside the country may be due to several factors, including climatic conditions, poverty level, nutritional status, socio-economic conditions, high population density, healthrelated behaviour, illiteracy and poor sanitation (20).
The infection with E. histolytica and G. lamblia was not signi cantly associated with gender (p > 0.05). However, males were 3.1 and 1.5 times more likely to test positive for E. histolytica and G. lamblia than females, respectively (COR = 3.10 and 1.46). This nding is in agreement with other reports that showed that more males were infected with both parasites (18,21,22). The majority of the positive cases were also reported in females, who also had higher odds of being infected with E. histolytica and G. lamblia (4). Other studies reported slight gender differences (1), while a signi cant association with boys being more infected than girls were also reported (23). These ndings indicate that the possibility of being infected with both parasites might be related to gender-speci c behaviour within a community. However, males are more exposed to outdoor activities, which make them more vulnerable to intestinal parasitic infections, whereas females mostly remain indoors.
This study revealed no signi cant differences in the prevalence of E. histolytica and G. lamblia associated with age. However, a relatively higher infection rate was found in children aged more than 10. These children were 1.6 times more likely to test positive for E. histolytica than those from the 6-9 years group. For G. lamblia, children in the same age were 52% less likely to be diagnosed positive than those aged 6-9 years (COR = 0.48, p-value = 0.130). This nding could be attributed to the common pattern of children behaviour, that is, their outdoor activities increase as they grow older, thereby increasing their exposure to intestinal parasites.
In conclusion: Urgent actions, i.e. long-term control measures and improvement of personal hygienic practices and sanitary and living conditions, must be implemented.

Limitations
The major limitation of this study was the small sample size. Therefore, the estimated prevalence cannot re ect the burden of the disease across the study area.