Molecular Copro-prevalence of Cryptosporidium in Egyptian Children and Evaluation of Three Diagnostic Methods

Research brief

Indian Pediatr 2014;51: 727-729

Molecular Copro-prevalence of Cryptosporidium in Egyptian Children and Evaluation of Three Diagnostic Methods

Mona M Fathy, *Noha M Abdelrazek, Fayza A Hassan and *Ayman A El-Badry

From the Departments of Clinical and Chemical Pathology, and *Medical Parasitology, Kasr Al-Ainy Faculty of Medicine, Cairo University, Cairo, Egypt.

Correspondence to: Dr Ayman A El-Badry, Medical Parasitology Department, Kasr Al-Ainy Faculty of Medicine, Cairo University, El Manial, Cairo, Egypt.
Email: [email protected]

Received: December 30, 2013;
Initial review: February 14, 2014;
Accepted: June 10, 2014.

Objective: To determine molecular prevalence of Cryptosporidium in a cohort of Egyptian children and compare three diagnostic tests. Methods: Stool samples from children with diarrhea (n=150) and from apparently healthy children (n=100) were examined for Cryptosporidium using microscopy, enzyme linked immuosorbant assay (ELISA) and nested polymerase chain reaction (nPCR). Results: nPCR detected Cryptosporidium in 22.4% of children. Acid–fast stain and ELISA showed false negativity but 100% specificity with nPCR as gold standard. Conclusion: Cryptosporidium is a common cause of diarrhea in children in Egypt.

Keywords: Diarrhea, Etiology, ELISA, Nested PCR.

Cryptosporidium is an emerging zoonotic pathogen with potentially life-threatening diarrhea in immuno-compromised patients[1]. Conventional methods for its identification include microscopic examination of its oocyst in fecal smears with acid-fast staining [2], and immunoassays that detect copro-antigen by ELISA. There are conflicting reports about the sensitivity of immuno-detection methods over microscopy [3]. Polymerase chain reaction (PCR) is being increasingly used for diagnosis [4]. The present study was designed to determine the molecular prevalence of Cryptosporidium in a cohort of Egyptian children, and to compare the utility of three diagnostic methods.

Methods

A total of 250 children (150 had diarrhea and 100 were non-diarrheal controls) attending the outpatient clinics of Abou El-Reesh Pediatric Teaching Cairo University Hospital, from February to July 2012, were enrolled in this cross-sectional study. The study was approved by Ethical Committee of Faculty of Medicine, Cairo University.

A single fecal sample from each child was collected and examined microscopically. Acid-fast staining was done from concentrated stool samples and examined for Cryptosporidium oocysts. Frozen fecal specimens were processed for fecal sandwich ELISA (RIDASCREEN Cryptosporidium-C1201- R-Biopharm AG, Germany) and nested-PCR (nPCR) assay. The nPCR was done by analysis of ssu rRNA gene. Copr-DNA was extracted, using Favor stool DNA spin columns isolation Mini Kit, incubated at 95ºC for one hour. Extracted DNA was amplified by nPCR [5] after adjusting annealing at 55oC. Laboratory procedures were performed in Clinical Chemistry Unit, Faculty of Medicine, Cairo University.

Diagnostics were evaluated and ranked by Multi-attribute utility theory [6] and Analytical hierarchy process [7] to help in decision-making, based on laboratory utility instead of being limited to the diagnostic yields. The six attributes were given a rank order from 1 to 3 and every attribute was prioritized by its importance over the other as per the laboratory’s infrastructure. The results were analyzed using the SPSS software, Version 15.0 (Chicago, IL, USA).

Results

Cryptosporidium copro-DNA and copro-antigen were detected in both diarrheal and non-diarrheal fecal samples, while Cryptosporidium oocysts were detected in diarrheal fecal samples only (Table I). Considering nPCR as the reference method, both acid-fast staining and ELISA had no false positivity (100% sensitivity and positive predictive value); however, they gave false negative results (32% and 55%, respectively). Using Multi-attribute utility, ELISA had the highest rank (2.39), followed by nPCR (1.96) and acid-Fast staining (1.73) (Table II).

TABLE I Diagnostic Yield of Fecal Acid-fast Stain, ELISA and nPCR in Detection of Cryptosporidium.

	ELISA	Microscopy
	+ve	–ve	+ve	–ve
PCR Positive
Diarrheal group (n=38)	22	16	18	20
Non-Diarrheal group (n=18)	9	9	0	18
PCR Negative
Diarrheal group (n=112)	0	112	0	112
Non-Diarrheal group (n=82)	0	82	0	82

TABLE II Multi-attribute Evaluation of Diagnostic Techniques for Cryptosporidium

Evaluation Items Priority value Acid-fast stain ELISA nPCR

Cost-effectiveness 0.95 3 2 1

Sensitivity 0.35 1 2 3

Specificity 0.35 3 3 3

Ease of use 0.9 1 2 3

Ease of interpretation 0.15 1 3 2

Ability to perform batch testing 0.5 1 3 2

Ability for species specification 0.07 0 0 3

Total* 1.73 2.39 1.96

*Priority values were multiplied by the ranks given for each attribute for every diagnostic technique.

Discussion

Our results demonstrated a high prevalence of Cryptosporidium in diarrheal and non-diarrheal fecal samples. Cryptosporidium were more frequent in children with diarrhea than in those with no diarrhea.

Our results were consistent with findings of other studies from developing world [8-11]. Although children without diarrhea are seldom tested for the presence of enteric pathogens, asymptomatic cryptosporidiosis may lead to growth retardation through mucosal damage causing nutrient malabsorption [12].

Many PCR primers using various target genes have been constructed to detect Cryptosporidium but 18 S rRNA (ssu-rRNA) gene loci are widely used [13]. PCR-inhibitors in feces and oocysts resistant to lysis lead to false-negative PCR results [14]. Despite these caveats, we obtained higher rates and this may be attributed to thermal treatment of samples during DNA extraction with elution of DNA in 2 steps using less amount of elution buffer. The wide use of PCR is hindered by its relative high cost and time taken to extract DNA from stool. However, PCR still holds importance for the identification of Cryptosporidium DNA in samples with low number of oocysts, and for detection of species.

We conclude that Cryptosporidium is an important enteric pathogen in Egyptian children. Immunodiagnosis of cryptosporidiosis may be of benefit, and molecular detection should also be an option. Cryptosporidiosis is underdiagnosed because clinicians fail to consider this diagnosis in immunocompetent patients with diarrheal illnesses (particularly children) and do not request stool analysis for Cryptosporidium.

Contributors: All authors contributed to study design, data collection, manuscript writing and its approval.

Funding: None; Competing interests: None stated.

What This Study Adds?

• Cryptosporidium is commonly isolated from fecal samples of Egyption children.

• Molecular methods and ELISA results in higher yield as compared to acid-fast staining.

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