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. 2021 Jan 27;45(2):319–323. doi: 10.1007/s12639-021-01349-2

Performance of formalin ethyl acetate, percoll sedimentation and ELISA for the detection of Cryptosporidium infection in asymptomatic children

Amal Farahat Allam 1,✉, Hend Aly El-Taweel 1, Hoda Fahmy Farag 1, Abdulsalam Farag Ibrahim Mohamed 2, Safia Saleh Khalil 1, Amel Youssef Shehab 1
PMCID: PMC8254688  PMID: 34295028

Abstract

This study aimed to assess the performance of formalin ethyl acetate (FEA)/modified Ziehl–Neelsen (MZN), and percoll technique/MZN for the diagnosis of cryptosporidiosis among asymptomatic children compared to ELISA coproantigen. The study was conducted on 100 children in a rural area in Kafr El-Sheikh governorate. Stool samples were collected and examined by the three techniques. Microscopic examination revealed the presence of acid-fast stained oocysts and non-acid fast ghost oocysts. The overall prevalence rate was 7% with an infection intensity of 1–5 oocysts/oil immersion field. FEA/MZN technique showed the highest diagnostic performance (5%) with 71.4% sensitivity and 98% negative predictive value (NPV) compared to the other techniques. ELISA revealed 3% prevalence, 42.9% sensitivity and 96% NPV. Percoll/MZN gave the lowest prevalence, sensitivity and NPV (1%, 14.29% and 93.9% respectively). Agreement fluctuated between moderate and poor regarding FEA/MZN versus ELISA and percoll/MZN versus both techniques. In conclusion, FEA/MZN gave the top diagnostic performance, yet it missed some positive cases. Its combination with ELISA coproantigen might prove beneficial for Cryptosporidium diagnosis. Percoll technique needs more validation by modifying the density gradient, speed of centrifugation, and staining methods.

Keywords: Cryptosporidium, Formalin ethyl acetate, Percoll, ELISA

Introduction

Cryptosporidiosis is a disease caused by a coccidian parasite belonging to the genus Cryptosporidium which has been recognized as one of the major agents of parasite induced diarrheal diseases worldwide (Rossle and Latif 2013). In children, mainly those living in developing countries, cryptosporidiosis can lead to malnutrition and developmental delays even in asymptomatic infections (Agnamey et al. 2010). Children, elderly people, and immunocompromised individuals are at risk of more severe infection (Mor et al. 2009; Zheng et al. 2018). Despite its universal occurrence, cryptosporidiosis is considered a neglected disease by the WHO, basically due to a shortage of studies in developing countries (Bamaiyi and Redhuan 2016). It is underdiagnosed as clinicians fail to consider its diagnosis in immunocompetent patients and accordingly do not bid stool analysis for Cryptosporidium (Clark 1999).

Conventional diagnostic modalities for Cryptosporidium identification include microscopic examination of modified Ziehl–Neelsen acid fast stain (MZN) of either fresh or preserved stool samples for the presence of oocysts and coproantigen detection using ELISA immunoassays (Morgan et al. 1998; Doganci et al. 2002).

Percoll, a technique used for the diagnosis of schistosomiasis was adopted in the present study for Cryptosporidium diagnosis (Allam et al. 2009). It is a density gradient medium widely used in separating cells, organelles, viruses, and other subcellular particles. In this technique, the particles are separated solely based on differences in density (Clavel et al. 1996; Entrala et al. 2000).

The present study aimed to assess the performance of microscopy-based techniques; FEA/MZN and percoll/MZN sedimentation compared to ELISA assay, in the diagnosis of Cryptosporidium infection among asymptomatic non diarrheic children.

Materials and methods

This study was carried out on 100 asymptomatic non diarrheic children randomly selected from those attending the local rural health unit in Arab El-Mahdar village, Kafr El-Sheikh governorate, Egypt. Their mean age was 5.19 ± 1.88 years, 54% were boys and 46% were girls. Ethical validation was obtained from the Ethics Board of the Medical Research Institute, Alexandria University. Collected stool samples of participating children were concentrated both by FEA (10% formalin preserved samples) and percoll (fresh samples) sedimentation techniques (Allam et al. 2009). The sediments obtained from both methods were stained by MZN and examined under oil immersion lenses for Cryptosporidium oocysts. The intensity of Cryptosporidium infection was quantified as the number of oocysts counted per microscopic field (under a 100× objective) of 50 μl volume of a concentrated stool sample. The scoring of infection intensity was as follows: low oocysts load+ (1–5 oocysts), moderate oocysts load++ (6–10 oocysts), high oocysts load+++ (11–15 oocysts), and very high oocysts load++++ (≥ 15 oocysts) (Matos et al. 2004; Smith 2008). A portion of each fecal specimen was tested with an ELISA assay for the detection of Cryptosporidium coproantigens using the commercial ELISA kit, RIDA SCREEN® Cryptosporidium (R-Biopharm).

Statistical analysis

Data collected were coded, tabulated, and analyzed using SPSS, Statistical Package for Social Science Software Version 20.0. Counts and percentages were used for describing and summarizing qualitative data. Arithmetic mean and standard deviation (SD) were used as measures of central tendency and dispersion respectively for normal distributed quantitative data. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of FEA/MZN, percoll/MZN and ELISA techniques were calculated against the “Gold” standard as summation of the combined results of the three techniques. Agreement between two qualitative variables was done by Cohen’s Kappa agreement test (at k < 0.2 poor agreement, k 0.2–0.4 fair agreement, k 0.41–0.6 moderate agreement, k 0.61–0.8 good agreement, k 0.81–1.0 very good agreement). In all tests, statistical significance was accepted at p < 0.05 (Ashby 1991).

Results

Microscopically, Cryptosporidium oocysts stained pinkish red by MZN appeared as thick-walled spherical structures of approximately 3–6 μm in diameter. Some non-acid fast Cryptosporidia appeared as oocyst ghosts (Fig. 1a, b).

Fig. 1.

Fig. 1

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Cryptosporidium oocyst stained pinkish red by MZN (a) and non-acid-fast Cryptosporidium oocyst ghost (b) (×1000)

The highest percentage of Cryptosporidium infection was detected by FEA/MZN (5%), followed by ELISA (3%) and percoll/MZN (1%) with an overall prevalence rate of 7%. All diagnosed cases were of low intensity (1–5 oocysts/oil immersion field).

Table 1 displayed the calculated parameters regarding the performance of the three employed techniques. FEA/MZN showed the highest sensitivity and accuracy (71.4% and 98% respectively) followed by ELISA (42.9% and 96% respectively). Percoll/MZN revealed the lowest performance.

Table 1.

Performance of the three used methods for the diagnosis of Cryptosporidium

Method Positive No (%) Sensitivity (%) (95% CI) Specificity (%) (95% CI) PPV (%) NPV (%) (95% CI) Accuracy (%) (95% CI)
FEA/MZN 5 71.43 (29–6.33) 100 (96.1–100) 100 97.89 (93.5–99.34) 98 (92.96–99.76)
Percoll/MZN 1 14.29 (0.36–57.87) 100 (93–100) 100 93.9 (91.97–95.45) 94 (87.40–97.77)
ELISA 3 42.86 (9.90–81.59) 100 (96.11–100) 100 95.88 (92.45–97.79) 96(90.07–98.90)
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PPV positive predictive value, NPV negative predictive value, CI confidence interval

Agreement analysis showed that five cases diagnosed positive by FEA/MZN were missed by percoll/MZN which only diagnosed one positive case missed by FEA/MZN, with a kappa index of 0.017 indicating poor agreement between both techniques.

Two Cryptosporidium infected cases gave concordant positive results both by ELISA and FEA/MZN techniques, however analysis of the discordant results revealed three cases detected only by FEA/MZN while one case positive by ELISA was missed by FEA/MZN. The inter-rater reliability concerning ELISA and FEA/MZN for the detection of Cryptosporidium was found to be moderate with a kappa index of 0.481 (p = 0.000), 95% CI (0.0429–0.915).

By analysis of the discordant results, three cases positive by ELISA were negative by percoll, while one case was missed by ELISA and diagnosed by percoll/MZN. Statistically, a poor agreement was noted between both techniques (k = 0.015) (Table 2).

Table 2.

Agreement between FEA/MZN, percoll/MZN, and ELISA techniques for the diagnosis of Cryptosporidium infection

FEA/MZN Percoll/MZN Total
Positive Negative
Positive 0 5 5
Negative 1 94 95
Total 1 99 100
k = 0.017 (poor agreement) p = 0.818 95% CI (0.0544–0.000370)
ELISA FEA/MZN Total
Positive Negative
Positive 2 1 3
Negative 3 94 97
Total 5 95 100
k = 0.481 (moderate agreement)  p = 0.000 95% CI (0.0429–0.915)
Percoll/MZN ELISA Total
Positive Negative
Positive 0 1 1
Negative 3 96 99
Total 3 97 100
k = 0.015 (poor agreement) p = 0.860 95% CI (0.0451–0.000380)
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Discussion

In the present study, FEA/MZN, percoll/MZN, and ELISA were evaluated for Cryptosporidium diagnosis in stool samples collected from asymptomatic children. The overall infection rate of Cryptosporidium infection was 7% as diagnosed by the three used methods. All cases diagnosed by microscopic examination were of low intensity. Variable results were reported in Egypt, the prevalence of Cryptosporidium in children ranged between 3.6 and 31.1% in different areas (Youssef et al. 2008; Abd El Kader et al. 2012). Elsewhere, the prevalence of Cryptosporidium infection ranged from 2.2% to 29% (Cama et al. 2008; Saleh et al. 2017). The variation of the disease prevalence in Egypt and in other areas may be explained by the different screened number of patients, personal hygiene, the used diagnostic methods, nutritional habits, socioeconomic and immunological status, differences in the environmental conditions and seasonal variations among different locations.

The present study revealed that, FEA/MZN technique showed the topmost diagnostic performance (5%), with high sensitivity and NPV compared to the other used methods. ELISA came second with 3% infected children. Percoll/MZN revealed the lowest infection rate of 1%, with 14.29% sensitivity and 93.9% NPV. Mittal et al. (2014) reported higher sensitivity of microscopic detection of preserved fecal samples stained with MZN compared to ELISA (100% versus 16%). On the contrary, Kaushik et al. (2008) and Gabr et al. (2014) concluded that ELISA is considered the best method for the detection of Cryptosporidium infection as its sensitivity amounted to 90% compared to microscopy (37–67%). This contradiction may be due to the fact that sedimentation methods are generally performed using low speed centrifugation. Given their small size and mass, cryptosporidial oocysts may become trapped in the ether or ethyl acetate plug and fail to sediment properly.

Despite the higher diagnostic yield of FEA/MZN compared to percoll sedimentation and ELISA, this technique failed to detect oocysts in two samples (one case tested positive by ELISA and one case was diagnosed by percoll/MZN). This could be explained by the occurrence of increasing numbers of non-acid-fast oocysts "ghosts “which may be due to the presence of a relatively high immunity level in immunocompetent children with Cryptosporidium infections (Utzinger et al. 2010). According to CDC report increasing number of non-acid-fast oocysts are present in resolving or recuperating cases. Such oocysts may not sediment as expected, giving rise to false negative results during microscopic examination (CDC 2019). Moreover, oocysts abundance can be low in immunocompetent persons which is our case (intensity 1–5/oil immersion field) who have sufficient immunity to downregulate oocysts production but insufficient immunity to downregulate asexual reproduction in enterocytes giving rise to ELISA positive results. In addition, oocysts are difficultly detected in concentrates made from formed fecal samples than from watery, diarrheal stool specimens (CDC 2019).

As for the agreement between the used methods, there was a moderate agreement between FEA/MZN and ELISA and poor agreement between FEA/MZN and percoll/MZN. The moderate agreement between ELISA and FEA/MZN is looking promising. Thus, a combination of both techniques can be considered a “gold standard” as sensitivity would be very high. Moreover, ELISA coproantigen has the major advantage of detecting asexual life cycle form of Cryptosporidium (Ungar 1990; Gabr et al. 2014). Discordant results and poor agreement were found between percoll/MZN and both FEA/MZN and ELISA. It detected only one positive sample which was missed by both techniques. Percoll technique may need more validation by changing the density gradient, raising the speed of centrifugation, and testing its compatibility with MZN compared to Giemsa stain.

In conclusion, the FEA/MZN method appears to be preferable to ELISA and percoll/MZN in terms of higher diagnostic yield. However, it is not sufficient to be used as a single diagnostic method for cryptosporidiosis as it missed some positive cases. Its combination with ELISA coproantigen may be beneficial for precise diagnosis of Cryptosporidium infection. Further studies on percoll are needed.

Acknowledgements

The authors wish to thank the participating children and their parents for their contribution in the present study.

Funding

No fund was obtained for the execution of the study.

Availability of data and materials

The data supporting the findings of this study are contained within the manuscript. The raw data are available by the corresponding author when requested.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

The study was approved by the Research Ethics Committee of the Medical Research Institute (MRI), Alexandria University.

Consent to participate

Informed consent was obtained from parents/guardians of participating children.

Consent for publication

All authors agree for publication.

Footnotes

Publisher's Note

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Associated Data

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Data Availability Statement

The data supporting the findings of this study are contained within the manuscript. The raw data are available by the corresponding author when requested.

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