Comparison of Assays for Sensitive and Reproducible Detection of Cell Culture-Infectious Cryptosporidium parvum and Cryptosporidium hominis in Drinking Water

Abstract

This study compared the three most commonly used assays for detecting Cryptosporidium sp. infections in cell culture: immunofluorescent antibody and microscopy assay (IFA), PCR targeting Cryptosporidium sp.-specific DNA, and reverse transcriptase PCR (RT-PCR) targeting Cryptosporidium sp.-specific mRNA. Monolayers of HCT-8 cells, grown in 8-well chamber slides or 96-well plates, were inoculated with a variety of viable and inactivated oocysts to assess assay performance. All assays detected infection with low doses of flow cytometry-enumerated Cryptosporidium parvum oocysts, including infection with one oocyst and three oocysts. All methods also detected infection with Cryptosporidium hominis. The RT-PCR assay, IFA, and PCR assay detected infection in 23%, 25%, and 51% of monolayers inoculated with three C. parvum oocysts and 10%, 9%, and 16% of monolayers inoculated with one oocyst, respectively. The PCR assay was the most sensitive, but it had the highest frequency of false positives with mock-infected cells and inactivated oocysts. IFA was the only infection detection assay that did not produce false positives with mock-infected monolayers. IFA was also the only assay that detected infections in all experiments with spiked oocysts recovered from Envirochek capsules following filtration of 1,000 liters of treated water. Consequently, cell culture with IFA detection is the most appropriate method for routine and sensitive detection of infectious Cryptosporidium parvum and Cryptosporidium hominis in drinking water.

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

“…In the case of the qPCR assay, background signal due to high inoculum densities is the major concern (Di Giovanni & LeChevallier, 2005). Hence, higher infection rates detected by the PCR assay may be the result of a relatively high frequency of false positives (Johnson et al, 2012). Additionally, growth supplements such as glucose and vitamins used in the inoculum for PCR assays have also been reported as major contributing factors in to increased C. parvum cell culture infection.…”

“…A comparison of RT-PCR and immunofluorescence assay (IFA) and microscopy assay (DAPI/PI) for testing viability suggested that RT-PCR is better for viability detection in Giardia, whereas IFA is superior for detecting viable Cryptosporidium oocysts (Johnson et al, 2012). Other molecular assays, such as FISH and nucleic acid probes, can be used to detect 18S rRNA in Giardia and Cryptosporidium.…”

“…The rationale behind selecting this assay includes: (i) a relatively low number of false positives with inactivated oocysts (one and three oocysts on monolayer); (ii) a relatively better performance than other assays with oocysts recovered from spiked filters using US EPA method 1623; (iii) it being one of the simplest methods with fewest processing steps; and (iv) its sensitivity to predict the number of infectious oocysts (Johnson et al, 2012;Bukhari & LeChevallier, 2003).…”

“…Recently, Johnson et al (2012) examined these assays for their relative sensitivity, reproducibility and frequency of false positives. Monolayers of HCT-8 cells were exposed to a variety of viable and inactivated oocysts to assess assay performance.…”

“…IFA has also been advocated for routine and sensitive detection of infectious C. parvum and C. hominis in drinking water. This suggestion was made on the basis of IFA assay efficiency to detect spiked infectious oocysts recovered from Envirocheck capsules (Pall Corporation) upon filtration of 1000 litres of treated water (Johnson et al, 2012;USEPA, 2005).…”