Real-Time PCR for Quantification of Giardia and Cryptosporidium in Environmental Water Samples and Sewage

Abstract

The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the ␤-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.

Novel Bumped Kinase Inhibitors Are Safe and Effective Therapeutics in the Calf Clinical Model for Cryptosporidiosis

“…The propagation of C. parvum (Iowa isolate) in calves and oocyst purification [34][35][36][37], synthesis of BKIs [22,25,27,38], in vitro CpCDPK1 enzyme assay [22,25,38], in vitro determination of C. parvum BKI sensitivity [22,25,38,39], neonatal mouse [36,40] and calf models of C. parvum infection [37,[41][42][43], and pharmacologic measurement of BKI plasma and stool levels and plasma protein binding have all been previously described [27,44]. BKI-1294 and BKI-1553 were synthesized on the pyrazolo [2,3-d] pyrimidine scaffold, while BKI-1517 was synthesized on a 5-aminopyrazole-4-carboxamide scaffold [25] (Figure 1).…”

Epidemiological survey in Łęczyńsko-Włodawskie Lake District of eastern Poland reveals new evidence of zoonotic potential of <i>Giardia intestinalis</i>

“…The diagnostics involve light microscopy examination of stool samples, ELISA or Direct Fluorescence Assay (DFA). However, only the molecular characterization of G.. intestinalis genotypes guarantees accurate identification of organisms and assessment of zoonotic transmission [5,6,7].…”

A Real-Time Polymerase Chain Reaction Assay for the Detection of the Myxozoan Parasite Henneguya Ictaluri in Channel Catfish

“…12,27 Quantitative real-time PCR has been used in the detection of pathogens in a number of aquatic systems, including fish tissues and environmental water samples. 1,3,[6][7][8][9][10]14 This assay is often more sensitive than conventional diagnostic techniques and offers the ability to quantify the pathogen, even at very low levels. In most cases, real-time PCR provides a rapid, more sensitive means of pathogen detection than conventional end-point PCR 13 and may be less subject to contamination than nested PCR assay.…”