Abstract
Quantitative real-time polymerase chain reaction (qPCR) assays to detect Cryptosporidium oocysts in clinical samples are increasingly being used to diagnose human cryptosporidiosis, but a parallel approach for detecting and identifying Cryptosporidium oocyst contamination in surface water sources has yet to be established for current drinking water quality monitoring practices. It has been proposed that Cryptosporidium qPCR-based assays could be used as viable alternatives to current microscopic-based detection methods to quantify levels of oocysts in drinking water sources; however, data on specificity, analytical sensitivity, and the ability to accurately quantify low levels of oocysts are limited. The purpose of this study was to provide a comprehensive evaluation of TaqMan-based qPCR assays, which were developed for either clinical or environmental investigations, for detecting Cryptosporidium oocyst contamination in water. Ten different qPCR assays, six previously published and four developed in this study were analyzed for specificity and analytical sensitivity. Specificity varied between all ten assays, and in one particular assay, which targeted the Cryptosporidium 18S rRNA gene, successfully detected all Cryptosporidium spp. tested, but also cross-amplified T. gondii, fungi, algae, and dinoflagellates. When evaluating the analytical sensitivity of these qPCR assays, results showed that eight of the assays could reliably detect ten flow-sorted oocysts in reagent water or environmental matrix. This study revealed that while a qPCR-based detection assay can be useful for detecting and differentiating different Cryptosporidium species in environmental samples, it cannot accurately measure low levels of oocysts that are typically found in drinking water sources.
Highlights
Cryptosporidium, the causative agent of cryptosporidiosis, is an intracellular apicomplexan parasite that infects the gastrointestinal tracts of animals and humans
In order to establish a Quantitative real-time polymerase chain reaction (qPCR) assay that could potentially detect all Cryptosporidium species and genotypes, two primer/probe sets were initially identified from the literature, JVA [24,29] and CRU18S [30], both originally designed for clinical investigations targeting the multi-copy 18S rRNA gene of Cryptosporidium
Specificity analyses of the CRU18S assay revealed that while no qPCR signals were detected from distantly related organisms including S. mansoni, E. coli, B. thuringiensis, B. cereus, S. flexneri, E. hellem, E. intestinalis, E. cuniculi, or closely related species like G. muris and G. duodenalis, significant qPCR signals were detected with T. gondii genomic DNA (gDNA) (16103 oocysts; CT values,30)
Summary
Cryptosporidium, the causative agent of cryptosporidiosis, is an intracellular apicomplexan parasite that infects the gastrointestinal tracts of animals and humans. Infection typically occurs through the fecal-oral route with waterborne transmission being most common. The disease is a self-limiting gastroenteritis but can potentially be fatal in immunocompromised individuals [1]. 26 Cryptosporidium species have been identified with dozens more genotypes reported [3,4,5,6,7,8,9]. Of the known species and genotypes reported, at least 12 have been found to infect humans, with Cryptosporidium parvum and Cryptosporidium hominis being associated with over 90% of the cryptosporidiosis outbreaks [5,6,7,10,11]
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