Introduction

Diarrhea remains a leading cause of morbidity and mortality among children under five years of age in sub-Saharan Africa, including Ethiopia [1]. Inadequate sanitation, limited access to clean water, and insufficient healthcare infrastructure contribute to the high prevalence of diarrheal diseases [2]. Diarrhea in children under five years of age can be caused by enteric viruses, bacteria, and parasites [3,4]. Among these, the intracellular protozoan parasite Cryptosporidium spp., which infects a wide range of mammalian species including humans, is a significant cause of gastroenteritis among young children [5,6]. Children living in resource-poor settings in sub-Saharan Africa and South Asia are particularly at risk of cryptosporidiosis [7].

Cryptosporidium spp. are transmitted through the fecal-oral route via the consumption of water or food contaminated with fecal matter, as well as by direct contact with infected individuals or animals [5]. It is highly infectious, and its transmission stage (the oocyst) is resistant to the disinfectants routinely used in the drinking water industry [8]. Globally, childhood cryptosporidiosis is estimated to cause approximately 44.8 million diarrheal episodes and 48,300 deaths annually, with the majority occurring in Africa [9]. Its burden is particularly high in sub-Saharan Africa, including Ethiopia, where Cryptosporidium infection is associated with prolonged diarrhea and a two- to three-fold increase in mortality among children with diarrhea [4,9].

Several factors may have a role in contributing to the high prevalence of Cryptosporidium infection, including the presence of infected individuals within households, contact with animals, open defecation practices, and inadequate breastfeeding [10,11]. However, the true magnitude of Cryptosporidium infections in low-income countries is likely underestimated due to the lack of systematic diagnostic testing for diarrheal disease etiologies and the widespread reliance on less-sensitive microscopic detection methods during routine clinical practice. Traditional diagnostic approaches, including microscopy with modified Ziehl-Neelsen staining, have limited sensitivity and require specialized expertise. Advanced techniques, like antigen-detection assays and polymerase chain reaction (PCR), offer improved sensitivity, but are not widely implemented in low-resource settings due to cost and technical constraints. This diagnostic gap contributes to an underestimation of the true prevalence and burden of Cryptosporidium infections in these regions [10,12].

Despite diarrhea being a significant health issue in Ethiopia [13,14], studies specifically focusing on Cryptosporidium infection remain limited. Existing studies on Cryptosporidium infection in Ethiopia show prevalence rates varying from 1% to 26% [15,16]. Most of this research has concentrated on individuals living with HIV, with relatively few studies involving school-aged or diarrheic children [16,17]. In addition, most of the previous studies used microscopic examination of stool smear using modified Ziehl-Neelsen staining, a method with a sensitivity of 55–75% and a specificity of 96–100% [10,18]. In contrast, stool samples stained with auramine-phenol (AP) and examined using light-emitting diode (LED) fluorescence microscopy have demonstrated superior diagnostic performance, with a sensitivity of 88% and a specificity of 99% [19]. Notably, there are no data on Cryptosporidium infection among young children in Eastern Ethiopia. Therefore, the present study aimed to assess the prevalence and factors associated with Cryptosporidium infection among children under five years of age with diarrhea in Eastern Ethiopia, using the light-emitting diode- auramine-phenol (LED-AP) method of diagnosis.

Methods

Study area

The study was conducted in six health centers, one from Shinile and five from the Dire Dawa Administration (DDA) (Fig 1). The study settings have two main seasons, a wet season from April to September and a dry season from October to March. Shinile is one of the woredas (districts) in the Somali region of Ethiopia, located at 9°40′N latitude and 41°50′E longitude. Its total population in 2022 was 70,181, of which 7,271 were children under five years of age (Shinile district report, 2022). The district has three health centers, but only one was fully operational and included in this study. Dire Dawa is located at 9°36′N latitude and 41°52′E longitude, approximately 515 km East of the capital, Addis Ababa, and the total population of DDA in 2022 was projected to be 535,684. Among the population, 192,106 individuals resided in rural kebeles, and 52,974 were children under five years of age [20]. DDA has two public hospitals and 14 health centers (Dire Dawa administration Regional Health Bureau report, 2022). For this study, five health centers (Gendakore, Goro, Jelobellina, Lagahare, and Melkajebdu) were randomly selected using a lottery method.

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Fig 1. Map of the study area in Eastern Ethiopia.

The map was constructed in ArcGIS 10.8.2 using district boundaries, https://gadm.org/download_country.html, and health facilities collected during the study. Data available under CC_BY 4.0 (Creative Commons Attribution, https://creativecommons.org/) license.

https://doi.org/10.1371/journal.pntd.0013386.g001

Results

Characteristics of study participants

A total of 756 children under five years of age, along with their caregivers, participated in this study. The male to female ratio was 1:0.8. The majority were from Lagahare and Goro health centers in Dire Dawa. A total of 548 (72.5%) and 176 (23.3%) of caregivers were the children’s mothers and fathers, respectively. Three hundred and two (40.0%) of the caregivers had no formal education. The majority (92.0%) reported having a latrine, of which 77.0% had a simple pit latrine (unimproved latrine). Around 30.0% of households owned at least one species of livestock. Among those who owned livestock, the majority (87.0%) had goats, while 51.0% had cattle. Moreover, 29.0% of households also had pet animals (dogs and cats). The majority (55.0%) of the children included were males. The children’s ages ranged from one to fifty-nine months (mean± SD = 20.8 ± 13.4; median = 16 months), and approximately 70% were under two years old (Table 1).

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Table 1. Sociodemographic characteristics of study participants and prevalence of Cryptosporidium infection in Eastern Ethiopia (n = 756).

https://doi.org/10.1371/journal.pntd.0013386.t001

Prevalence of Cryptosporidium infections

Overall, 15.2% (115/756; 95% CI: 12.7–17.9) of the children enrolled in the study were positive for Cryptosporidium by LED-AP microscopy. Of the 115 Cryptosporidium-positive samples, 41 (35.6%), 46 (40%), and 28 (24.4%) had oocyst loads categorized as 1 + , 2 + , and 3 + , respectively. The proportion of Cryptosporidium-positive samples was similar in each of the study health centers, ranging from 12.1% to 18.9%; in Jelobelina health center the proportion was higher (25.0%), but this was not statistically significant (Table 1).

There was no significantly higher occurrence of Cryptosporidium in any of the four age groups being compared (Table 1). There was a significantly higher occurrence of Cryptosporidium infection among children who were not exclusively breastfed. The highest prevalence of Cryptosporidium infection was detected in June (28.5%), followed by July (26.6%) and September (24.3%) (Fig 2). Moreover, Cryptosporidium infection was detected in 24.2% of children with diarrhea lasting more than three days before seeking medical care and 18.0% of those presenting with fever.

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Fig 2. Association between prevalence of Cryptosporidium infection among children under five years old with diarrhea and average rainfall (mm) of the study area.

https://doi.org/10.1371/journal.pntd.0013386.g002

Factors associated with Cryptosporidium infection

Results obtained in our bivariate and multivariable analyses are presented in Supporting information (S1 Table) and Table 2, respectively. The variables “age” and “season” were included in the final model regardless of the p-values obtained in the bivariate analysis, assuming them to be potential confounders for many of the factors.

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Table 2. Factors associated with Cryptosporidium infections among diarrheic children under five years of age attending health centers in Eastern Ethiopia, 2023 (n = 756).

https://doi.org/10.1371/journal.pntd.0013386.t002

In the final model, Cryptosporidium infections were nearly twice as likely to occur during the wet season than the dry season. Children whose caregivers had no formal education were 2.4 times more likely to be infected with Cryptosporidium than children whose caregivers had a diploma or higher educational status. Children living in households with a family member reporting diarrhea in the two weeks prior to sampling were two times more likely to be infected with Cryptosporidium compared to children in households without recent diarrhea cases. Similarly, children who had contact with individuals experiencing diarrhea were 1.7 times more likely to be infected with Cryptosporidium compared to their counterparts (Table 2).

Caregivers that did not wash their hands after toileting were nearly three times more likely to have children infected with Cryptosporidium than those who practiced handwashing. Similarly, Caregivers who used toilet paper were 1.6 times more likely to have children infected with Cryptosporidium than those did not use it. This means using toilet paper alone is not a hygienic intervention unless paired with proper handwashing, safe disposal, and clean sanitation practices. Children with three or more older siblings were two times more likely to be infected with Cryptosporidium compared to those with no older siblings. Children who were not exclusively breastfed were 1.8 times more likely to be infected than their exclusively breastfed counterparts (Table 2).

Discussion

This study found that the prevalence of Cryptosporidium infection among diarrheic children under five years old seeking healthcare in Eastern Ethiopia was 15.2% (95% CI: 12.7–17.9). Season, caregivers’ educational status, childbirth order, exclusive breastfeeding, household history of diarrhea, contact with diarrheic individuals, handwashing after toileting, and the use of toilet paper were factors significantly associated with an increased risk of Cryptosporidium infection.

The prevalence of Cryptosporidium infection in the current study is consistent with some of those reported in similar studies conducted among children with diarrhea attending healthcare facilities in Ethiopia and other sub-Saharan African countries (S1 Table). Cryptosporidium prevalence rates of 12.8% was documented in diarrheic pediatric population in Bahir Dar, Ethiopia [24], 12.9% in Gabon [25], 13.9% in Mozambique [26], and 15.0% and 15.6% in Tanzania [21,27]. However, the prevalence found in this study is higher than previous reports from Ethiopia such as 9.0% in Jimma [23]. It is also higher than the prevalence reported in a sub-study conducted among 58 symptomatic children under three years of age attending health facilities in Mekelle, which found a prevalence of 5.2% [28]. Similarly, the prevalence observed in this study is higher than reports from other parts of Africa which reported 11.0% in Ghana and 12.0% in Madagascar [21] 8.4% and 11.0% in Kenya [29,30], 9.1% in Malawi [31], 3.4 and 11.0% in Mozambique [32,33], 4.0% in Nigeria [34] and 6.0% in South Africa [35]. In contrast, the prevalence found in this study was lower than those reported in some other African studies (S2 Table) including rates of 30.0% in Angola [36], 20.5% in Botswana [6], 21.0% in Gabon [21], 18.4% in Guinea-Bissau [37], 23.5% and 32.0% in Kenya [17,38], 22.8% and 23.1% in Nigeria [39,40] and 27.1% in Sudan [41]. These discrepant figures may be due to differences in local environmental conditions, seasonal variations, nutritional habits, personal hygiene practices, sanitation, access to safe drinking water, sewage water management, and methodological factors such as sample size, sampling techniques and diagnostic procedures. Moreover, the higher prevalence observed in this study compared to some previous studies that used conventional microscopy examination may be attributed to the use of more sensitive and specific LED-AP fluorescence microscopy in the current study.

In this study, there was a statistically significant association between season and the prevalence of Cryptosporidium infection, which was nearly two times more likely to occur during the wet season than during the dry season, with the highest prevalence rate (28.6%) being detected in June. Similar findings have been reported in previous studies. For instance, cryptosporidiosis cases peaked during the hottest months in Pakistan [42], and during the period April-June in Kenya [30]. These findings may be due to the fact that children tend to gather and play together more frequently during this season, thereby increasing their contact with surface waters potentially contaminated with Cryptosporidium oocysts. Additionally, in the study area, there is a common practice of storing water for future use. Furthermore, the area is prone to flooding during the rainy season, which may contaminate rivers, streams, and stored water sources with fecal matter. These water sources are used for cattle feeding, irrigation, and drinking, increasing the risk of Cryptosporidium infection among residents.

Caregivers’ educational status was significantly associated with Cryptosporidium infection in children, with a higher likelihood of infection among children whose caregivers had no formal education. Similarly, previous studies conducted in Cameroon [43], Ghana [44] and Mozambique [26] have found that a lack of secondary or higher education was associated with an increased risk of Cryptosporidium infection. Another study conducted in Mozambique reported that children of illiterate caregivers were more likely to be infected with Cryptosporidium [45]. This is likely because education is associated with improved hygiene practices, sanitation conditions, feeding habits, handwashing behaviors, and breastfeeding duration. Moreover, more educated parents may have higher incomes, enabling them to afford hygiene products such as soap and bottled water for drinking and food preparation for their children.

The current study showed that children with three or more older siblings were two times more likely to be infected with Cryptosporidium compared to those with no older siblings. This finding is consistent with a study conducted in India reporting the presence of an older sibling in the household as a significant risk factor for cryptosporidiosis [46]. Additionally, an increase in the number of siblings living at home in Peru has been associated with higher odds of Cryptosporidium infection [47]. This may be because overcrowding conditions facilitate closer and more frequent personal contacts, enhancing the transmission risk of Cryptosporidium through the fecal-oral route [48]. Furthermore, older siblings, who may have lower hygiene standards than adults, are often involved in the care of younger siblings, potentially increasing the risk of infection.

Breast milk provides effective passive immunity to newborns against a wide range of enteric pathogens [49]. Children who were not exclusively breastfed were 1.6 times more likely to develop Cryptosporidium infection than those who were exclusively breastfed. This finding is consistent with a study conducted in south east of Iran that reported breastfed infants were less susceptible to Cryptosporidium infection [50]. Similarly, a study in Cameroon found a higher prevalence of Cryptosporidium infection among children who were not exclusively breastfed compared to those who were [43]. Other studies have also reported that non-exclusively breastfed children are more likely to develop diarrhea [51,52]. This protective effect of breastfeeding is attributed to the presence of secretory immunoglobulin A in breast milk, which helps shield children from mucosal pathogens. Additionally, breast milk contains other protective molecules such as IgG, IgM, IgD, lactoferrin, lactoperoxidase, and various leukocytes that safeguard the mucous membrane against gastrointestinal pathogens [53]. Moreover, children who are not exclusively breastfed may be more exposed to Cryptosporidium through contaminated food and drinks.

We found that diarrhea in the household and contact with diarrheic individuals were significant risk factors for Cryptosporidium infection among diarrheic children (p-value < 0.05). Similarly, previous studies have indicated that household diarrhea is one of the most relevant risk factors associated with Cryptosporidium infection in low- and middle-income countries [11]. Additionally, contact with a person with diarrhea has been identified as a risk factor for Cryptosporidium infection. This implies that close person-to-person contact is a major transmission route for Cryptosporidium, consistent with findings from other studies [21,54].

Furthermore, this study showed that not washing hands after using the toilet and using toilet paper or were significant risk factors for Cryptosporidium infection in children. This may be because individuals who use toilet paper perceive themselves as clean, and therefore often neglect thorough washing of their hands. This can lead to contamination of food, surfaces, or direct contact with children, facilitating fecal-oral transmission of Cryptosporidium. The used toilet paper may also contaminate the environment around the toilet, and children may acquire the infection from that environment or from family members. Supporting this, a study found that infrequent handwashing practices were an independent predictor of increased diarrheal morbidity [55]. Taken together, these findings highlight the importance of case contact in transmitting Cryptosporidium, similar to other fecal-orally infectious diseases, and emphasize the role of personal hygiene such as proper handwashing and child hygiene in reducing Cryptosporidium transmission.

In this study, Cryptosporidium infection was more prevalent in children who had diarrhea for more than three days before the study (p-value < 0.05). This finding is consistent with studies from other sub-Saharan African countries, which have shown that Cryptosporidium infection is associated with prolonged or persistent diarrhea [4,9,29]. Moreover, fever was commonly observed in children with Cryptosporidium infection in this study. However, other studies have reported no significant association between fever and Cryptosporidium infection [48,56].

In the present study, age and gender of the child, treatment of drinking water, availability of a latrine, and handwashing before feeding and after helping a child defecate showed no significant association with Cryptosporidium infection (p > 0.05). Previous studies have also indicated that the prevalence of cryptosporidiosis was not statistically associated with age [43] and gender, with a p-value of >0.05 [42,57]. This may be because children are likely exposed to the same risk factors, regardless of their sex [58]. However, a study conducted in Tigray (north Ethiopia) showed that Cryptosporidium infection was related to age, with higher prevalence in younger age groups (≤ 15 years), and to sex, with a higher prevalence in females [28]. This discrepancy may be due to the broader age range in the Tigray study, which included individuals aged 4–80 years, whereas the current study focused on children aged 1–59 months. Unlike young children, adult females and males may not be equally exposed to the same risk factors for Cryptosporidium infection, and there may also be differences in their immune responses.

In contrast to the well-established role of animals in the transmission of cryptosporidiosis in some settings [59], as well as findings from a systematic review and meta-analysis in low- and middle-income countries [11] and in Ethiopia [16], which indicated that animal contact was significantly associated with Cryptosporidium infection, this study found that the presence and contact with animals were not significantly associated with cryptosporidiosis among children. This finding is consistent with a previous study in Ethiopia and elsewhere [60,61]. This may be due to the fact that zoonotic transmission of Cryptosporidium occurs relatively rarely in Sub-Saharan Africa compared with in developed countries like in Europe, predominantly due to the Cryptosporidium species distribution being very different [62,63]. In our study also, where livestock ownership was not associated with Cryptosporidium infection, anthroponotic species, particularly C. hominis, predominated in the study area [64], as has previously been observed in other urban and semi-urban settings of developing countries [62]. In addition, among the of Cryptosporidium species detected from livestock fecal samples investigated during the study period, only C. ubiquitum, C. xiaoi and C. ryanae were identified, of which only C. ubiquitum is considered to have zoonotic potential [64].

This study has strengths, such as utilizing a more sensitive and specific laboratory test, AP fluorescence microscopy, to diagnose Cryptosporidium infection, unlike most previous studies that relied on the modified Ziehl-Neelsen staining method. As a result, this study provides a relatively more accurate estimate of the infection burden. Additionally, data were collected over a one-year period, which is important for assessing seasonal variations in Cryptosporidium infection among diarrheic children under five in eastern Ethiopia. The study is further strengthened by the molecular characterization of Cryptosporidium species from both children and livestock [64]. It also has some limitations, such as the cross-sectional design, which assesses variables at a single point in time, making it challenging to infer cause-and-effect relationships. Additionally, there may be recall bias, as caregivers may provide incorrectly recalled information about symptoms or risk factors. Only a single stool sample was tested per child, potentially leading to false negatives, as Cryptosporidium oocysts may shed inconsistently. Therefore, the findings of this study should be interpreted with caution.

Conclusions

Cryptosporidium spp. infection was found to be highly prevalent in diarrheic children under five years of age seeking healthcare in Eastern Ethiopia. Several factors were significantly associated with the infection, including hygiene parameters in the child’s environment, exclusive breastfeeding status, diarrhea in the household, seasonal effects, and caretakers’ educational status. Therefore, raising community awareness about the seasonality of disease, the importance of exclusive breast feeding, and the relevance of proximity and person to person close contact for transmission of Cryptosporidium infection is recommended. Molecular-based studies that investigate the frequency and diversity of Cryptosporidium species and genotypes would also be valuable for better understanding the sources of transmission in Ethiopia.

Supporting information

Acknowledgments

We sincerely thank AHRI for supporting for fieldwork and laboratory analysis. We also extend our gratitude to Haramaya University for its logistical support, including transportation facilitation for data collection and other study-related activities. We are deeply grateful to all the children and their caregivers for their participation in this study, as well as to the health center staff for their valuable assistance in sample and data collection. Additionally, we appreciate the contributions of the laboratory technologists from the respective health centers and the School of Medical Laboratory Sciences, Haramaya University. We also acknowledge the drivers who assisted during data and sample collection. We extend our thanks to the Oromia Regional Laboratory for providing auramine O stains, the Dire Dawa Regional Laboratory for providing phenol chemicals, and the Sabata Veterinary Institute for providing potassium dichromate. Finally, we express our gratitude to the One Health staff at AHRI, Dr. Ashenafi G. Giorgis for facilitating support from AHRI, Mr. Lema Demise for his support in statistical analysis, and Mr. Galano Mamo for drawing the study area map.