Immunomagnetic Separation (IMS)-Fluorescent Antibody Detection and IMS-PCR Detection of Seeded Cryptosporidium parvum Oocysts in Natural Waters and Their Limitations

Abstract

Detection and enumeration of Cryptosporidium parvum in both treated and untreated waters are important to facilitate prevention of future cryptosporidiosis incidents. Immunomagnetic separation (IMS)-fluorescent antibody (FA) detection and IMS-PCR detection efficiencies were evaluated in two natural waters seeded with nominal seed doses of 5, 10, and 15 oocysts. IMS-FA detected oocysts at concentrations at or below the three nominal oocyst seed doses, illustrating that IMS-FA is sensitive enough to detect low oocyst numbers. However, the species of the oocysts could not be determined with this technique. IMS-PCR, targeting the 18S rRNA gene in this study, yielded positive amplification for 17 of the 18 seeded water samples, and the amplicons were subjected to restriction fragment length polymorphism digestion and DNA sequencing for species identification. Interestingly, the two unseeded, natural water samples were also PCR positive; one amplicon was the same base pair size as the C. parvum amplicon, and the other amplicon was larger. These two amplified products were determined to be derived from DNA of Cryptosporidium muris and a dinoflagellate. These IMS-PCR results illustrate that (i) IMS-PCR is able to detect low oocyst numbers in natural waters, (ii) PCR amplification alone is not confirmatory for detection of target DNA when environmental samples are used, (ii) PCR primers, especially those designed against the rRNA gene region, need to be evaluated for specificity with organisms closely related to the target organism, and (iv) environmental amplicons should be subjected to appropriate species-specific confirmatory techniques.Cryptosporidium parvum, an intestinal protozoan parasite, continues to be an important cause of waterborne gastrointestinal disease worldwide. Due to oocyst robustness consisting of environmental stability and resistance to normal water disinfection processes (14,22), the low infectious dose (10), and the lack of chemotherapy (23), detection and enumeration of this organism in both treated and untreated waters have become a focus of the water industry in order to prevent future incidents of cryptosporidiosis.The currently accepted technique for oocyst recovery from water samples is immunomagnetic separation (IMS)-fluorescent antibody (FA) detection, as described by United States Environmental Protection Agency (EPA) Method 1623 (25). The reported IMS-FA recovery rates for oocysts seeded into previously concentrated water pellets of various turbidities have been 62 to 100% (oocyst seed density as determined by dilution, 36 to 976) (20), 55.9 to 83.1% (oocyst seed density as determined by dilution, 89.1 to 98.7) (17), 68 to 83% (oocyst seed density as determined by dilution, 525 to 870) (3), and 84.3% (oocyst seed density as determined by flow cytometry, 100) (19). In an additional study, in which oocysts were seeded into 10-liter grab samples of various turbidities, the reported recoveries ranged from Ͻ1.7 to 56.6% (oocyst seed densities, 1,615 and 2,880) (8).However, the criteria for o...

Development of Procedures for Direct Extraction of Cryptosporidium DNA from Water Concentrates and for Relief of PCR Inhibitors

“…Reduction or elimination of PCR inhibitors prior to, during, or after DNA extraction has become an important step in molecular diagnosis of microbial pathogens in water and other environmental samples. Currently, pathogen isolation by IMS and culture enrichment prior to DNA extraction are standard procedures to eliminate or reduce PCR inhibitors (9,18,23,27,31,34,35). These, however, become impractical for organisms that have no IMS procedures or that cannot be cultured.…”

“…Three basic approaches have been used in general to overcome the effect of PCR inhibitors, including purification of microorganisms prior to DNA extraction, removal of inhibitors during or after DNA extraction, and relief or suppression of the effect of PCR inhibitors when performing PCR. In the detection of Cryptosporidium, the most commonly used method is the purification of the oocysts by IMS prior to DNA extraction (9,12,18,23,27,31,35), which effectively eliminates or greatly reduces the substances that might be inhibitory to PCR amplification. However, IMS is expensive, and its performance is affected by the type of commercial kits used, pH, and dissociation procedures (7,16,30).…”

Detection and Discrimination of Cryptosporidium parvum and C. hominis in Water Samples by Immunomagnetic Separation-PCR

“…IMS-FA, however, is not able to discriminate Cryptosporidium species because IMS and FA use the genus-specific antibody for the capture and detection of the oocysts, respectively (19). Thus, we developed the nested PCR-RFLP method for the highly divergent Cpgp40/15 gene to detect the C. parvum and C. hominis genes and discriminate between the two species.…”

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.…”

“…The protozoan parasite Cryptosporidium parvum has recently been recognized as an important cause of waterborne gastrointestinal disease worldwide (Fayer, et al, 2000;Sturbaum, et al, 2002). The organism can cause self-limited diarrhea in immunocompetent hosts and chronic, life threatening diarrhea in immunocompromised individuals, such as patients receiving immunosuppressive therapy and those suffered from acquired immunodeficiency syndrome (AIDS) (Fayer and Ungar, 1986).…”