Cell Culture-Taqman PCR Assay for Evaluation of Cryptosporidium parvum Disinfection

Abstract

Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log 10 units) with LP-UV (20 mJ/cm 2 ) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log 10 unit being inactivated. These results demonstrate the inability of MIOX to inactivate Cryptosporidium. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water.Cryptosporidium is recognized as a frequent cause of waterborne disease in humans (3). The disease has been documented worldwide, with speculation that many undiagnosed cases of gastroenteritis may have been caused by this parasite (21,38). Two means of transmission have been shown to be contaminated drinking water in distribution systems (25,28) and swimming pools (17). Cryptosporidium oocysts are resistant to standard chlorine-based disinfection procedures that are a key barrier in the transmission of other waterborne pathogens in potable water. Although some water treatment processes remove oocysts, any oocysts that break through represent a potential threat to human health due to their relative chlorine resistance. Various methods of disinfection have been investigated by a number of research groups; these methods include UV light (4, 10, 11, 13, 14, 23, 35), ozone (5, 18, 22, 29), chlorine dioxide (8,12,22,30,34), mixed oxidants (MIOX) (6, 40), and chlorine (7,22,17). Many disinfection studies have used animal infectivity or surrogate in vitro assays to determine the viability of oocysts after disinfection. Animal bioassays are considered the "gold standard" for assessing Cryptosporidium oocyst infectivity, and the neonatal mouse model has been used extensively in the assessment of oocyst disinfection. However, this model has limited applications for the assessment of waterborne oocysts because type 1 (human) Cryptosporidium parvum cannot infect mice. The human genotype can be cultured in gnotobiotic pigs (41), and this model has been used to assess drug efficacy (37).Significant developments in determining oocyst infectivity have included cell culture (CC) assays for type 1 oocysts (20) and for type 2 oocysts (14,15,20,31,36,39). These methods have used culturing of oocysts in HCT-8 (human ileocecal adenocarcinoma) cells. Evaluation of these methods has shown the CC assay with the HCT-8 cell line to be equivalent to the gold standard neonatal mouse infectivity assay (32,35). Shim et al. (35) demonstrated that CC assays provide a level of sensitivity similar to t...

Inactivation of Single-Celled Ascaris suum Eggs by Low-Pressure UV Radiation

“…However, it is important to point out that during Ascaris egg development to the third-stage larva, the Ascaris egg grows from one cell to about 600 cells (35), which is significantly different from what happens in the life cycle of the single-celled prokaryotic protozoans Giardia and Cryptosporidium. For protozoans exposed to UV, infectivity studies or cell cultures are necessary to measure viability, because the DNA is not fully replicated until the organisms reproduce, after infection (16,18,33,38). In Ascaris, however, the complete DNA of the organism is replicated when the single cell in the egg undergoes the first two cell divisions before it differentiates into the germ and somatic cell lines that form the larva (26).…”

Aged HCT-8 Cell Monolayers Support Cryptosporidium parvum Infection

“…In addition to the study of Cryptosporidium biology, cell culture methods have been used to study Cryptosporidium inactivation by disinfectants (4,13,23,26,28), efficacy of chemotherapeutic agents (3,5,34,37), and detection of infectious oocysts in water samples (1,7,10,17). The developmental stages of the parasite in cell culture are detected using a variety of techniques, including immunofluorescent assay microscopy (27), flow cytometry (31), reverse transcription-PCR (22), and quantitative PCR (5,8,11,15).…”

Quantitative-PCR Assessment of Cryptosporidium parvum Cell Culture Infection

“…Many C. parvum cell culture studies have used 48 h postinoculation as the endpoint of infection (16,24,26,28,29,31). In this study, CC-QSD revealed that infection at 48 h postinoculation was only 60% of the amount of infection at 72 h postinoculation.…”

“…Therefore, the CC-QSD standard curve generated by linear regression of mean background-subtracted CC-QSD C T values plotted against the log number of AHBSS-trypsin-treated oocysts inoculated per cell monolayer was sufficient. A similar relative quantitation approach using a DNA standard curve generated with only oocyst DNA, instead of infected cell monolayers, was recently reported for a disinfection study (16). The development of a universal standard curve for the absolute quantitation of infective C. parvum oocysts is a significant challenge.…”