Introduction

Cryptosporidium possesses 24 valid species and more than 44 genotypes infecting many vertebrates; including humans and animals, which differ significantly in their molecular signatures (Cama et al. 2008). Thirteen cases of intestinal and gastric Cryptosporidium species infecting immunocompetent and immunocompromised humans have been reported until now (C. parvum, C. hominis, C. meleagridis, C. felis, C. canis, C. suis, C. muris, C. andersoni, C. fayeri, C. cuniculus, C. ubiquitum, and C. viatorum) (Fayer et al. 2010; Elwin et al. 2012). Among them, C. parvum (which infects not only humans but also ruminants and perhaps a few other animals) and C. hominis (which is almost exclusively a human parasite) are the most common species involved in clinical infections (Sulaiman et al. 2005).

Cryptosporidium is listed as a neglected disease by the World Health Organization, largely due to a lack of studies in developing countries, but is now gaining increasing attention (Savioli et al. 2006).

Because of the limitations in the specific detection of Cryptosporidium using microscopic, immunological, and/or flowcytometric methods, a wide range of nucleic acid-based methods had been developed and evaluated for the identification of species, as well as the detection of genetic variation within and among species of Cryptosporidium with a high diagnostic yield (Smith et al. 2006).

The current study aimed to determine the prevalence of Cryptosporidium among a cohort of Egyptian children targeting the Hsp90 gene and RFLP analysis for species identification. Due to the lack of existent studies assessing risk factors in Egypt, this study was designed to investigate the role of collected data variables for susceptibility to Cryptosporidium infection among individuals.

Methods

Sampling and data collection

A cross-sectional study was designed involving 431 stool samples (331 diarrheic and 100 apparently healthy non-diarrheic children) collected from children of both sexes ranging in age from 1 to 12 years, presenting with diarrhea and/or other GIT symptoms recruited from the outpatient clinic in Abu El Rish pediatric hospital, Kasr Al-Ainy School of Medicine, and Cairo University from April 2013 to January 2014. Data included the recording of their sociodemographic and environmental data. Parents of young children provided consent forms and responded to the questionnaire.

Extraction of the genomic DNA

Genomic DNA was extracted using Favor Prep stool DNA isolation Mini Kit (Favorgen Biotech corporation ping-Tung 908, Taiwan, Cat. No. FASTI001) with modification in the form of prolongation of incubation to 95 °C for 1 h after thermal shock (cycling of deep freezing in liquid nitrogen for 5 min and immediately transferred into a water bath 95 °C for 5 min; repeated for 5 cycles) then the purified DNA was measured for concentration and purity.

Copro-nPCR/RFLP analysis

The nPCR was done by analysis of Hsp90 gene. Amplification of 835–844 for the primary reaction and a fragment of 676–685 for the secondary reaction, using two sets of oligonuclutide primers (Table 1) with the reaction components and the cycling conditions carried out according to Feng et al. (2009) with modification in the form of 12.5 μl master mix, 200 nM from each primer, and 3 μl of the template DNA for the primary reaction and 1 μl for the secondary one in a total volume of 25 μl and 50 °C annealing temperature for the primary and the secondary reactions. The amplified products were visualized with 1.5% agarose gel electrophoresis after ethidium bromide staining. The amplified products of nPCR of positive samples were digested using StyI and HphI endonuclease (Table 2) according to manufacturer’s instruction and resolved, using 3% metaphor electrophoresis after ethidium bromide staining.

Table 1 The used primers and their sequences (Feng et al. 2009)
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Table 2 Hsp90 RFLP (restriction enzymes) (Feng et al. 2009)
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Statistical analysis

Data were statistically analyzed using the statistical package SPSS version 17 (Chicago, IL, USA). Data were analyzed with Fisher᾿s exact test and multiple logistic regression. All variables that were significantly associated with the Cryptosporidium prevalence in the univariate model were included in a multivariate logistic regression.

Results

Out of 431 examined stool samples with nPCR, 84 (19.5%) stool samples were Cryptosporidium positive (78 diarrheic and 6 non-diarrheic); among them 75 (89.3%) were C. hominis, 6 (7.1%) were C. parvum, and 3 (3.6%) were non-typed. Their relative data were tabulated in Table 3.

Table 3 Data of Hsp90 positive cases
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Among the studied variables, only diarrhea, the type of water, and stool consistency were significantly associated (P˂0.05) with detection of Cryptosporidium. After these variables were subjected to a multivariate analysis using logistic regression, there was an estimated increase in the risk of Cryptosporidium among children suffering from diarrhea 4.8 times greater than the non-diarrheic children, with soft and liquid stools, 6.5 and 3.3 times, respectively, relative to formed stool, and 4.4 times for children drinking tap water relative to mineral water (Table 4).

Table 4 Multivariate analysis for nPCR Cryptosporidium-positive cases
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In contrast, none of the studied variables, including gender distribution, age distribution, animal contact habits, and gross features of stool showed any significant association among genotypes (P > 0.05) (Table 4).

Discussion

Cryptosporidium is a highly prevailing parasite among studied diarrheic children for about a quarter of them (23.6%) using nPCR, with an overall prevalence of 19.5% in both groups. There is a difference in the reported prevalence of Cryptosporidium in Egypt in the last decades; most of the studies report a high molecular prevalence of up to a quarter of examined patients (El-Settawy and Fathy 2012; Fathy et al. 2014; Ghallab et al. 2016), however, much lower results (4.6%) were also reported in Egypt using PCR (Abd El-Kader et al. 2011) and these studies claimed that the presence of fecal inhibitors and contaminants such as bilirubin, bile salts, and others can inhibit DNA amplification, yielding less accurate detection results (Abd El-Kader et al. 2011).

Until now, few studies in Egypt have addressed the Cryptosporidium transmission dynamics in humans, despite it being pivotal in terms of establishing effective prevention and disease control policies. In the present study, C. hominis, an anthroponotic Cryptosporidium species, was significantly more prevalent (89.3%) than C. parvum (7.1%). Mixed infections were not detected, indicating that the main source of Cryptosporidium infection in the study group was of human rather than zoonotic source. Akiyoshi et al. (2003) reported that in mixed infections, C. parvum predominates and rapidly displaces C. hominis which adds more proof that in our study, it is a C. hominis predominance and may explain the non-typed species we obtained. Our results are in concordance with those of only one study (Abd El-Kader et al. 2011) and contradictory to those of other Egyptian studies and most studies from other Middle Eastern countries (Eida et al. 2009; Al-Brikan et al. 2008; Hijjawi et al. 2010; Iqbal et al. 2011). Our data and the data of Abd El-Kader et al. (2011) suggest a clear anthroponotic transmission; this discrepancy in the results may be explained by the fact that Abd El-Kader et al. (2011) and our study were conducted in Great Cairo, while the contrasting results were found in more rural parts of Egypt.

This predominance of C. hominis is in accordance with molecular studies in Australia, Canada, Japan, USA, and developing countries (Xiao and Fayer 2008). In contrast, studies in the UK have shown C. parvum predominance (McLauchlin et al. 1999, 2000).

Among studied variables, diarrhea, the type of water, and stool consistency showed statistically significant association with cryptosporidiosis. Our results were in agreement with those of other reports in Mexico and Iran, in which there were no reported significant differences between cases with cryptosporidial diarrhea and age, race, ethnicity, or sex (Nair et al. 2008; Saneian et al. 2010; Bushen et al. 2007). It was reported that C. hominis is contracted at an earlier age than C. parvum. Samie et al. (2006) reported that C. hominis was equally distributed between males and females. GIT symptoms, including diarrhea, abdominal pain, flatulence, itching, vomiting, and/or appetite loss (Chauret et al. 1999), animal contact (Hijjawi et al. 2010), and type of water supply (Goh et al. 2004; Beach 2008; Garvey and McKeown 2009; Al-Warid et al. 2012) are well-known risk factors for cryptosporidiosis.

The occurrence of clinical manifestations in Cryptosporidium can be related in part to the different Cryptosporidium species and subtypes of C. hominis. Cama et al. (2008) reported that C. hominis was associated with diarrhea, vomiting, nausea, and general malaise, while C. parvum, C. canis, C. felis, and C. meleagridis were associated with diarrhea only. Environmental, clinical, and host behavioral factors may act as important risk factors for Cryptosporidium infection, susceptibility, or disease prevalence, but do not affect the pathogenicity or the course of the disease (Mumtaz et al. 2010).

In the present study, besides the studied variables that may induce susceptibility of cryptosporidiosis in Egypt, there are added variables such as higher population densities with a greater chance of person-to-person transmission and variation in socioeconomic status within the same geographical areas. This might also explain the discrepancies in the reported results for the prevalence of the disease between past studies and our current research findings.

Conclusion

Cryptosporidium is an indigenous, prevailing intestinal parasite among children in Cairo with a predominance of C.hominis, suggesting anthroponotic transmission rather than zoonotic. The significant majority of Cryptosporidium infections were detected in diarrheic patients, who relied upon tap water sources; their stool was soft and yielded more parasites than the patients with liquid stool. Due to the absence of any effective curative treatments, determining the prevailing species of Cryptosporidium and identifying the significant etiological risk factors are essential in order to identify its transmission dynamics and plan preventive and control strategies.