Detection of cryptosporidia and Cryptosporidium parvum oocysts in environmental water samples by immunomagnetic separation-polymerase chain reaction

Abstract

Cryptosporidium parvum has emerged as one of the most important new contaminants found in drinking water. Current protocols for the detection of cryptosporidia are time-consuming and rather inef®cient. We recently described an immunomagnetic separation±polymerase chain reaction (IMS±PCR) assay permitting highly sensitive detection of C. parvum oocysts in drinking water samples. In this study, a second IMS±PCR assay to detect all cryptosporidial oocysts was developed, and both IMS±PCR assays were optimized on river water samples. A comparative study of the two IMS±PCR assays and the classical detection method based on an immuno¯uorescence assay (IFA) was carried out on 50 environmental samples. Whatever the type of water sample, the discrepancy in C. parvum detection between the IFA and IMS±PCR took the form of IFA-negative/IMS±PCR-positive results, and was caused mainly by the greater sensitivity of IMS±PCR as compared with IFA. Of the 50 water samples, only ®ve tested positive for C. parvum using IMS±PCR, and could constitute a threat to human health. These results show that both IMS±PCR assays provide a rapid (1 d) and sensitive means of screening environmental water samples for the presence of cryptosporidia and C. parvum oocysts.

Evaluation of QIAamp DNA mini kit for removing of inhibitors in detection of Cryptosporidium parvum oocysts in water samples by a nested-PCR assay

“…Although immunomagnetic separation (IMS) is now more commonly used and many researches have been used the method along with PCR for detection of Cryptosporidium oocysts in environmental samples (Hallier-Soulier and Guillot, 1996;Hallier-Soulier and Guillot, 2000;Rimhanen-finne, et al, 2001;Sturbaum, et al, 2002;Xiao, et al, 2001), but the procedure selectively separate the Cryptosporidium oocysts. However it would be advantageous to perform parallel detection assay for multiple pathogen on a single sample.…”

Cryptosporidiosis in Animals and Man: 2. Diagnosis

“…Using PCR, 1-10 5 oocysts have been detected. Compared with IF microscopy, the PCR has shown to increase detection sensitivity 10-10 4 fold in environmental and fecal samples in some studies (Hallier-Soulier and Guillot, 2000;Lowery et al, 2000). A twostep nested-PCR protocol was used to amplify a fragment of the SSU-rRNA gene of Cryptosporidium (~830 bp) and compared with IFA in detection of Cryptosporidium in ewes and lambs (Santin et al, 2007).…”

Potential molecular tools for assessing the public health risk associated with waterborne Cryptosporidium oocysts

“…The most prominent molecular genotyping methods used for determining the public health significance of oocysts are as follows: (i) nested PCR to detect Cryptosporidium oocyst wall protein (COWP) (Pedraza-Díaz et al, 2001); (ii) immunomagnetic separation (IMS)-PCR assay, which permits the highly sensitive detection of C. parvum oocysts in drinking water samples (Hallier-Soulier & Guillot, 2000); (iii) PCR-RFLP assay to amplify Cryptosporidium parvum oocyst wall protein (COWP) for rapid detection (Leone et al, 2009); (iv) FISH technique using fluorescently labelled oligonucleotide probe (18S rRNA Cry1 probe) for detection of C. parvum oocysts in water samples (Alagappan et al, 2009); and (v) hsp70 and 18S rRNA multiplex PCR, which only permits the differentiation of C. parvum and C. hominis (Di . These genotyping methods have been applied with promising results to the host-specificitybased source tracking of Cryptosporidium in watersheds (Jenkins et al, 2010).…”