Assessment of the potential occurrence of Cryptosporidium species in various water sources in Sharqia Governorate, Egypt

Abstract

Cryptosporidium species are enteric apicomplexan parasites associated with diarrhoeal disease in humans and animals globally. Waterborne outbreaks resulting from contamination with the infective oocysts are common worldwide. Updated reports on waterborne protozoal infections are needed to identify emerging pathogens and susceptible populations. Therefore, this study aimed to assess the current profile of Cryptosporidium contamination of various water sources in Sharqia Governorate, Northeastern Egypt. For this purpose, eighty samples were collected from five different water types (canal, tap, tank, filtered, and groundwater), distributed in four major cities (El-Hessenia, Fakous, Zagazig, and Belbies) in Sharqia Governorate. All water samples were examined using conventional microscopy, ELISA, and real-time PCR (RT-PCR) techniques. Based on microscopic analysis, the Cryptosporidium protozoan was identified in 25% of the tested water samples. The RT-PCR assay has allowed for the quantification of Cryptosporidium oocysts in different types of water. Canal water exhibited the highest Cryptosporidium contamination levels (mean = 85.15 oocysts/L), followed by water tanks (mean = 12.031 oocysts/L). The study also provided a comparative evaluation of ELISA and RT-PCR for the diagnosis of Cryptosporidium infection. RT-PCR performed better than ELISA in terms of analytical accuracy (97.50% vs. 86.25%) and specificity (100% vs. 83.33%). However, ELISA showed a higher sensitivity (95.00% vs. 90.00%) for Cryptosporidium recovery. Our findings could serve as a platform for further investigations into the potential risks associated with water contamination in Sharqia Governorate.

Supplementary information: The online version contains supplementary material available at 10.1007/s12639-024-01675-1.

Keywords: Cryptosporidium; Detection; Egypt; Identification; Prevalence; Waterborne parasites.

Fig. 1

Map of the study area: Sharqia Governorate is situated in Northeastern Egypt. Water samples for the study were collected from four different cities, namely, El-Hessenia, Fakous, Zagazig, and Belbies (d-maps.com)

Fig. 2

Flowchart outlining the conducted filtration steps. a Collected water sample in a clean plastic container. b The first sample filtration using a mesh sieve to remove coarse particles. c The second filtration through a nitrocellulose membrane filter, which is folded twice with the upper surface facing out. d Elution of the membrane filter with phosphate-buffered saline (PBS) in a 50-ml centrifugation tube. e The sample is next centrifuged at 6000 g for 10 min. f The supernatant is discarded, and the recovered sediments are collected and preserved in potassium dichromate (2.5%)

Fig. 3

Standard curve of the Real-time PCR (RT-PCR) assay. The curve was generated using a tenfold serial dilutions of Cryptosporidium genomic DNA template purified from a known concentration of Cryptosporidium oocysts (1 × 10⁶ oocysts/ml).The (Ct) values were plotted in the Y-axis against the logarithm of the absolute amount of DNA copies on the X-axis. The linear regression equation of the standard curve is (y) = − 3.4686 + 39.107, with a correlation coefficient (R²) of 0.9941 and amplification efficiency (E) of 94.25%

Fig. 4

Photomicrographs depicting the oocysts of the coccidian elements captured during light microscopic examination. The slides were prepared using temporary and permanent staining measures. a Cryptosporidium oocyst in an unstained water sample (black arrow, X400). b Cyclospora oocyst (black arrow), with visible sporozoites inside the oocyst (red arrow) (unstained smear, X400). c Cryptosporidium oocyst in an iodine stained sample (black arrow, X1000). d Cyclospora oocyst (black arrow) (Iodine stain, X1000). e Cryptosporidium oocysts after using the temporary eosin stain (black arrows, X1000). f Cyclospora oocyst (black arrow) (MZN stain, X1000)

Fig. 5

Results of microscopic identification, ELISA, and real-time PCR (RT-PCR) in the detection of Cryptosporidium infection in different water samples

Fig. 6

Analytical sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and accuracy of ELISA and real-time PCR for detecting Cryptosporidium infection in water samples. The diagnostic results of both ELISA and RT-PCR measures were evaluated using conventional microscopy as a gold standard