Prevalence of diarrhea causing protozoan infections and associated risk factors in diarrheic under five children in Bahir Dar town, northwest Ethiopia: pediatric clinic based study

Diarrheal diseases cause about 3 million deaths annually, mainly to children under five years of age. This study determined the prevalence of diarrhea causing intestinal protozoans and the associated risk factors in diarrheic children aged below five years in Bahir Dar town, Ethiopia. From November 2011 to February 2012, pediatric clinic based study was conducted. Stool specimens were collected from 422 diarrheic children using labeled stool cups and examined microscopically for the presence of cysts, trophozoites, and oocysts. Direct saline, formalin-ether concentration, and modified Ziehl-Neelson staining methods were used for stool examination. Eighty six (20.4%) of the stool samples had one or more intestinal protozoan parasites. Prevalence of Entamoeba histolytica/dispar was 15.2% (64/422). Cryptosporidium spp., and Giardia intestinalis were detected as single infections in 16 (3.8%), and four (0.9%) of the study subjects, respectively. A relatively high prevalence of protozoal infection was recorded and therefore public health education about diarrhea causing protozoans and potential risk factors for diarrhea should be given to caretakers/parents for prevention and control of diarrhea under five children.


INTRODUCTION
Diarrhea remains a leading cause of mortality among children under five years of age around the world (Black et al., 2010). Globally, 1.3 billion cases of acute diarrhea occur in children below five years annually, of which more than 3 million cases are fatal; 80% of these deaths occurred in children below two years of age (Samal et al., 2008). The WHO (2004) ranks diarrheal disease as the second most common cause of morbidity and mortality in children of the developing world following acute respiratory infections.
In developing countries, diarrheal disease kills 1.5 million children under the age of five years every year (Elias, 2008). In Africa each child experiences five episodes of diarrhea per year and 800,000 children die each year from diarrhea *Corresponding author:endtg2002@yahoo.com infected by E. histolytica/dispar and G. intestinalis, respectively (Getamesay Mulatu et al., 2015).
Exposure to diarrhea causing pathogens is conditioned by factors like age of the child, quality and quantity of water, availability of toilet facilities, housing conditions, level of education, household economic status, place of residence, feeding practices, and the general sanitary conditions (personal or domestic hygiene) around the house (Stephen, 2005). It is, therefore, very important to determine the interaction of these factors in relation to child's survival. However, information on the prevalence of these protozoans and the associated risk factors in children living in Bahir Dar town is limited.
Hence, the present study was undertaken to determine the prevalence of diarrhea causing intestinal protozoan infections and associated risk factors in under five children presenting with diarrheal disease.

Study area, design and period
Pediatric clinics based study on diarrhea causing

Sample size determination and sampling technique
The sample size (n) was calculated using statistical formula of sample size calculation, n=Z 2 P (1-P)/d 2 (Danile, 1999).
Where, n=the minimum sample size required, Z=standard score corresponds to 1.96 at 95% confidence interval, d=is margin of sampling error tolerated (5% marginal error was used), p=prevalence of intestinal protozoa. Since the overall prevalence of intestinal protozoa in the specific study subjects in the study area was unknown, a prevalence of 50% was considered for the calculation of sample size and a sample size of 384 was obtained. To minimize errors from the likelihood of non-compliance, 10% of the sample size was added and a final sample size of 422 was considered in the study.
All diarrheic children aged below five years who visited the only two private pediatric clinics (Arsema and Universal Pediatric Clinics) in Bahir Dar town and who were not on anti-protozoal drugs within fifteen days before the commencement of stool specimen collection were selected for study.
The diarrheic children were distinguished from non-diarrheic children based on the passage of unusually consistent loose or watery stools, usually at least three times in a 24 hour period (WHO, 2005).

Data collection and laboratory analysis
A questionnaire was developed based on known risk factors and to fill the questionnaire caretakers/ mothers were interviewed by medical doctor and nurse during clinical examination. The questionnaires included socio-demographic, environmental and behavioral factors that expose the children to protozoal infection.
After proper instruction of caretakers about stool collection, about 2g of fresh stool was collected from each child. Every specimen was checked for its label, quantity, and procedure of collection. The collected stool specimens were immediately transported to Bahir Dar University Biomedical Sciences Research Laboratory. Stool examinations were performed using direct saline and formol-ether concentration technique by a trained laboratory technician (WHO, 2000). For the detection of Cryptosporidium spp., Modified Ziehl-Neelsen method was used (WHO, 2003). To ensure validity of the slide test, all positive slides and random sample of some of the negative slides were re-examined by another experienced laboratory technician who was blind to the diagnosis of the first slide-reader.

Data management and analysis
Data were entered and analyzed using SPSS version 16 software. Initially the association between exposure and the presence of infection was assessed using Chi-square test. Crude Odds Ratio (COR) was then determined using univariate logis- P-values less than 0.05 were considered to be statistically significant.

Ethical considerations
The study was reviewed and approved by the Ethical Review Board of Bahir Dar University.
Official letter was submitted to Arsema and Universal Pediatric Clinics. Both informed verbal and written consent were sought from the mothers/ caretakers of each diarrheic child. The purpose of the study was explained and sufficient time was given for the interviewees to decide whether to participate in the study or not. The positive cases got treatment from the clinics.

Socio-demographic characteristics
A total of 422 diarrheic children were included for examination. Of these, 239 (56.6%) were males    where a higher proportion of children whose feeding materials were washed less frequently experienced diarrhea compared to children whose feeding utensils were washed frequently (AOR: 6.57; 95% CI: 3.63-118.00; p-value 0.005) ( Table 6).

Prevalence of intestinal protozoal infection
Regular medical checkup of the child's caretaker was also associated with the risk of infection by protozoa. Children whose caretakers undergo regular medical checkup were less likely to be infected compared to those children whose caretakers who did not undergo medical checkup (AOR: 48.67; Availability of toilet, and type of water source (untreated surface water versus bottled water) showed significant associations in the univariate analysis, but their significance disappeared in the final step of the multivariate analysis (p>0.05) ( Table 6).
From the behavioral factors entered in the first step of multivariate analysis, using complementary food, duration of breast feeding, and bottle feeding practice remained significant in the final step of the analysis (

DISCUSSION
The overall prevalence of protozoa infection in the present study was 20.4%. This figure is lower than the prevalence reported in children under five in Ghana (38%) (Osumanu, 2010). The prevalence in the study area was higher when compared with the prevalence reported from Vietnam (11.3%) (Takanashi et al., 2009) (Ali et al., 2003), but it is quiet lower when compared with the prevalence of Entamoeba in Nigeria (65.7%) (Ogunlesi et al., 2006).   (Frost et al., 2000). But, it is much lower when compared with the prevalence reported from Shiraz, Iran (21.4%) (Nahrevanian and Assmar, 2008) and Nigeria (19.9%) (Dozie et al., 2011). The low prevalence of Cryptosporidium spp. may be due to the intermittent nature of excretion of oocysts by the parasite (Navin and Juranek, 1984). It is possible that the infection rate observed in the present study would have been even higher, if more than a single stool specimen was collected from each child.
In the case of G. intestinalis, the prevalence in the present study (0.9%) was comparable with the prevalence conducted in children in Italy (0.8%) (Guidetti et al., 2010) and  cho, 2006) and Ghana (Boadi and Kuitunen, 2003).
This could be because educated mothers practice good hygiene, better child feeding and weaning practices and the interpretation of symptoms and timely action to childhood illness, all of which increase a child's resistance against infectious diseases (Frost et al., 2005).
Child's age was found to be a significant predictor of diarrheal morbidity due to protozoa in children in the age category of 12-23 months. This is might be because of the combined effect of the low level of maternal acquired immunity, the introduction of food or liquid contaminated with pathogens, and direct contact with human and animal excreta when the child starts to crawl (Usfar et al., 2010). On the contrary, children in the age category of 48-59 months and above are less vulnerable to diarrhea because of the protective effects of a relatively prolonged breastfeeding and the less exposure to contaminated agents (Piechulek et al., 2003).
The likelihood of transmitting diarrhea in those caretakers who were reluctant in having regular medical checkup for their health even when they look healthy was very high. This is because usually caregivers of the children who look asymptomatic may harbor numerous parasites whether they have diarrhea or not and as a result, the chance of transmitting the infection is very high (Young and Briscoe, 1987). For example, in the case of E. histolytica/dispa,in up to 90% of the infections, the symptoms are absent or very mild and the infected individuals serve as unidentified carriers and may become responsible for transmission (Gatti et al., 2002).
Children whose caretakers who did not wash their hands with soap after visiting toilet were more infected by intestinal protozoans compared to children whose caretakers washed their hands with soap after visiting toilet. The result is consistent with a study conducted in Turkish school children (Ostan et al., 2007). It is clear that washing hands with soap reduces the infectious organisms present on the hands thereby reducing diarrheal episode by 31% (Ejemot et al., 2008). Other similar studies have also shown that washing hands with soap regularly has reduced the risk of intestinal diseases in children (UNICEF/WHO, 2009).
Whenever there is a family member who was infected by protozoa in the same house, it was shown that it is a significant predictor of diarrheal morbidity in children. In other study it has been shown that an eight fold increase in the odds of catching diarrhea due to protozoa in children. This shows that a higher probability of exposing children to infectious agents from food handlers of the family and childcare providers (Medeiros et al., 2004).
Moreover, the health of the child may be compromised if the food items/bottles are easily contaminated by flies.
Higher proportion of children whose feeding materials were washed less frequently experienced diarrhea than infants whose feeding utensils were washed frequently. This is because less frequently washed infants' feeding materials are more susceptible to contamination by flies, cockroaches, and hands of the caretakers. So, frequent washing of the child's feeding material reduces the infectious organism present on it and this observation agrees with the findings that were reported by Holt et al. (2007).
Breastfeeding protects children from infection particularly in the first three months of life (Butte et al., 2002). This is because breast milk contains oligosaccharides, immunoglobulin-A (IgA), and lactoferrin that have been shown to protect the child from gastro-enteritis (Hanson and Korotkova, 2002). In addition, the use of breast milk also offers fewer opportunities for the infant not to become infected from other foods and water (PATH, 2008).
The present study showed that children who were not breastfed in the first six months were at higher risk of developing diarrhea.
Exclusive breast feeding reduces exposure of the child to contaminated foods, drinking water, and feeding utensils. According to WHO (2001), the optimal duration of exclusive breastfeeding is very important for the first six months. In present study it was revealed that those infants who were not exclusively breastfed in the first six months of age were more infected by protozoans compared to infants who were exclusively breastfed. The present finding is consistent with findings reported from India (Dabasmita and Bratati, 2005) and Belarus (Kramer et al., 2003).
The introduction of other liquids or foods marks the beginning of a child's exposure to possibly contaminated foods and liquids (Dewey et al., 2001). Ideally, complementary foods should be introduced at age of six months and breastfeeding should continue for up to two years or even longer to increase birth intervals (WHO, 2003).
The result of multivariate analysis showed that the risk of developing diarrhea due to protozoa was fifteen times higher in children who started complementary feeding before the age of six months than those children who started after six months. This could be because infant who were weaned too early will lack antibodies and nutrients supplied by breast milk, which could expose them to different parasitic infections (Dewey et al.,

2001).
It has been documented that there is a high chance of contamination and greater risk of diarrhea associated with bottle feeding (Holt et al., 2007).
In this study, bottle feeding was significantly associated with intestinal protozoal infection.
This is because bottles are highly susceptible and exposed for contamination by flies and fecally contaminated hands (Holt et al., 2007  25-30. Frost, F.J., Muller, T and Craun, G. (2000). Serological analysis of a cryptosporidiosis epidemic. International Journal of Epidemiology 29: