Dataset Information

The Applicability of TaqMan-Based Quantitative Real-Time PCR Assays for Detecting and Enumerating Cryptosporidium spp. Oocysts in the Environment.

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.

SUBMITTER: Staggs SE

PROVIDER: S-EPMC3689768 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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The Applicability of TaqMan-Based Quantitative Real-Time PCR Assays for Detecting and Enumerating Cryptosporidium spp. Oocysts in the Environment.

Staggs Sarah E SE Beckman Erin M EM Keely Scott P SP Mackwan Reena R Ware Michael W MW Moyer Alan P AP Ferretti James A JA Sayed Abu A Xiao Lihua L Villegas Eric N EN

PloS one 20130621 6

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 ...[more]

PMID: 23805235

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Project description:BackgroudCryptosporidium species are zoonotic protozoan parasites responsible for gastroenteritis in various animals and humans. The diagnosis of Cryptosporidium presents many challenges. This research attempted to match the diagnostic efficiency of the modified Ziehl-Neelsen technique (mZN), immunochromatographic assays (IC), and enzyme-linked immunosorbent assay (ELISA) for the detection of Cryptosporidium in faecal samples of cattle in Kuwait. In addition, polymerase chain reaction (PCR) was utilised to determine the predominant species infecting cattle in Kuwait and correlating the detected species with the results of different diagnostic tests used, the presence or absence of clinical signs, and the age group of the infected cattle.ResultsOf 400 analysed faecal samples, Cryptosporidium positive samples were 23%, 15.25%, and 14% using IC, ELISA, and mZN. IC had the highest sensitivity (74.07%), and mZN had the highest specificity (98.29%) using a composite reference standard (CRS) as a gold standard. The rapid IC test results in high false-positive results of cryptosporidiosis, whereas using mZN alone is insufficient to declare a negative faecal sample. Only 74.5% (35/47) of Cryptosporidium-positive samples by the three assays could be amplified by PCR. This study was the first to genotype Cryptosporidium in Kuwait. Cryptosporidium parvum (n = 26) was the dominant species detected from cattle samples, followed by C. andersoni (n = 6), C. bovis (n = 2), and C. raynae (n = 1). The findings showed a statistically relevant relationship between diarrhoea and the detection of Cryptosporidium spp. in faecal samples of cattle (p-value = 0.0003). Pre-weaned calves were the most vulnerable age group to Cryptosporidium spp. infection (p-value = 0.0007).ConclusionFor screening of Cryptosporidium infection in faecal samples, antigen detection or PCR methods combined with one of the microscopy techniques should be used. Cryptosporidium parvum was the prepoderant Cryptosporidium spp. recovered from cattle samples in Kuwait followed by C. andersoni. Cryptosporidium parvum is a significant risk factor for diarrhoea in pre-weaned calves. However, further study is needed as many other causes of diarrhoea in calves must be ruled out before a diagnosis of Cryptosporidium diarrhoea can be made.

Dataset Information

The Applicability of TaqMan-Based Quantitative Real-Time PCR Assays for Detecting and Enumerating Cryptosporidium spp. Oocysts in the Environment.

Publications

The Applicability of TaqMan-Based Quantitative Real-Time PCR Assays for Detecting and Enumerating Cryptosporidium spp. Oocysts in the Environment.

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