Detection of Infectious <i>Cryptosporidium</i> in Filtered Drinking Water

Aboytes, Ramon; Giovanni, George di; Abrams, Felicia A.; Rheinecker, Corey; McElroy, W.J.; Shaw, Nancy J.; LeChevallier, Mark W.

doi:10.1002/j.1551-8833.2004.tb10706.x

Cited by 53 publications

(46 citation statements)

References 22 publications

(32 reference statements)

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“…The disease cryptosporidiosis is usually a self-limiting diarrhea in healthy adults but may be life threatening for persons with weakened immune systems. Cryptosporidium oocysts are ubiquitous in environmental waters, are resistant to chlorination, and have been found in treated drinking water (1,18).…”

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confidence: 99%

“…The use of cell culture to study the intracellular development of Cryptosporidium was described over 2 decades ago (6). 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).…”

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Aged HCT-8 Cell Monolayers Support Cryptosporidium parvum Infection

Sifuentes

Giovanni

2007

Appl Environ Microbiol

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Cell culture assays in various formats have been used to study the infectivity of Cryptosporidium spp. as well as to determine the infectivity of naturally occurring oocysts in water. Currently, cell culture assays for infectious Cryptosporidium spp. in water have largely been limited to practice in research laboratories. One obstacle to the routine use of Cryptosporidium cell culture assays for the analysis of water samples is the coordination of water sample collection and processing with readiness of cell culture monolayers. For most Cryptosporidium cell culture assays, monolayers are allowed to develop for 24 to 48 h to reach 80 to 100% confluence prior to inoculation. In this study, we used immunofluorescent assay microscopy to evaluate freshly confluent (2-day-old) and aged (8-to 67-day-old) HCT-8 cell monolayers for their ability to support Cryptosporidium parvum infection. HCT-8 monolayers as old as 67 days were clearly shown to support infection. In two of three experiments, aged monolayers (8-to 11-day-old and 11-to 22-day-old, respectively) developed the same number of C. parvum clusters of infection as freshly confluent monolayers. Results suggest that it may be possible to use cell monolayers from freshly confluent to 3 weeks old on hand for infectivity assays without having to schedule sample processing to coincide with development of freshly confluent monolayers. This would make Cryptosporidium cell culture assays much more feasible for water quality and utility laboratories.

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Aged HCT-8 Cell Monolayers Support Cryptosporidium parvum Infection

Sifuentes

Giovanni

2007

Appl Environ Microbiol

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“…Therefore, incubation periods may have an effect on the ability to detect infection, particularly with very low numbers of infectious oocysts. In our previous studies using conventional CC-PCR to assay environmental samples, an incubation period of 72 h was used, which may have aided the detection of low numbers of naturally occurring C. parvum oocysts (1,7,19). Therefore, on the basis of our successful CC-PCR analysis of environmental water samples and the CC-QSD data presented here, we recommend a 72-h incubation period for the CC-QSD method.…”

Section: Discussionmentioning

confidence: 96%

Quantitative-PCR Assessment of Cryptosporidium parvum Cell Culture Infection

Giovanni

LeChevallier

2005

Appl Environ Microbiol

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A quantitative TaqMan PCR method was developed for assessing the Cryptosporidium parvum infection of in vitro cultivated human ileocecal adenocarcinoma (HCT-8) cell cultures. This method, termed cell culture quantitative sequence detection (CC-QSD), has numerous applications, several of which are presented. CC-QSD was used to investigate parasite infection in cell culture over time, the effects of oocyst treatment on infectivity and infectivity assessment of different C. parvum isolates. CC-QSD revealed that cell culture infection at 24 and 48 h postinoculation was approximately 20 and 60%, respectively, of the endpoint 72-h postinoculation infection. Evaluation of three different lots of C. parvum Iowa isolate oocysts revealed that the mean infection of 0.1 N HCl-treated oocysts was only 36% of the infection obtained with oocysts treated with acidified Hanks' balanced salt solution containing 1% trypsin. CC-QSD comparison of the C. parvum Iowa and TAMU isolates revealed significantly higher levels of infection for the TAMU isolate, which agrees with and supports previous human, animal, and cell culture studies. CC-QSD has the potential to aid in the optimization of Cryptosporidium cell culture methods and facilitate quantitative evaluation of cell culture infectivity experiments.Ongoing challenges for the water industry are Cryptosporidium detection and infectivity determination. Mouse models have frequently been used to determine the infectivity of Cryptosporidium oocysts (2,10,18,20). However, animal infectivity determination is costly and labor-intensive and requires as long as 2 weeks to complete. Furthermore, it is not feasible for the analysis of environmental samples. In an attempt to simplify infectivity determination, several in vitro methods have been developed, such as in vitro excystation and microscopic assays evaluating the uptake or exclusion of fluorogenic dyes (3, 4) and fluorescence in situ hybridization techniques (35). Various PCR strategies have also been developed to assess the viability of Cryptosporidium parvum oocysts, including the integration of in vitro excystation (5, 9, 36) and the use of reverse transcription-PCR for the detection of mRNA transcripts found only in viable oocysts (24,30).Strategies using in vitro cell culture of C. parvum are attractive alternatives to animal infectivity testing. In most of these assays, oocysts are stimulated to excyst by exposure to sodium hypochlorite (bleach), bile enzymes, and/or acidic pH (7,23,24,32) and are then inoculated onto in vitro-cultivated cell culture monolayers. Sporozoites released from oocysts invade and replicate within the intracellular environment of the cells. After incubation, parasites are detected within the cell cultures by a variety of techniques, including immunofluorescence microscopy (28), reverse transcription-PCR (24), and standard PCR (7). Quantification of cell culture infection for pharmaceutical and disinfection studies has been achieved with an enzyme-linked immunosorbent assay (38). Most-probablenumber format...

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“…Turbidity and particle number removal was lower than oocysts removal. Aboytes et al (1), however, investigating 82 conventional (rapid) filters, found no differences in water quality (turbidity and microbial indicators) between sites with and without infectious oocysts in filtered waters.…”

Section: Introductionmentioning

confidence: 92%

Association between the concentration of protozoa and surrogates in effluents of the slow sand filtration for water treatment

Heller¹,

Vieira²,

Brito³

et al. 2007

Braz. J. Microbiol.

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Currently, a major challenge on producing high-quality drinking water is to monitor pathogens, such as Giardia, Cryptosporidium and enteric viruses. Due to limitations of the analytical methods available to detect pathogens in water, the research of surrogate indicators is an up-to-date subject. In view of these aspects, a pilot scale study was performed to evaluate the association between microbiological and physical indicators and the presence of Giardia spp and Cryptosporidium sp in the effluent of upflow and downflow slow sand filters. The results showed that efficient bacterial removal could indicate suitable protozoa removal. Although coliforms and Escherichia coli do not present the appropriate physiological profile for an "ideal" indicator, they are still good references for drinking water microbiological quality, specifically for slow sand filtration. The results also point out to the need of deeper researches about the use of anaerobic spores as routine indicator. Regarding the control of Cryptosporidium outbreaks, the expectation that a single indicator will satisfy all purposes is unreal. It may be more useful to know the advantages and disadvantages of several indicators, and integrate them appropriately.

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Detection of Infectious Cryptosporidium in Filtered Drinking Water

Cited by 53 publications

References 22 publications

Aged HCT-8 Cell Monolayers Support Cryptosporidium parvum Infection

Aged HCT-8 Cell Monolayers Support Cryptosporidium parvum Infection

Quantitative-PCR Assessment of Cryptosporidium parvum Cell Culture Infection

Association between the concentration of protozoa and surrogates in effluents of the slow sand filtration for water treatment

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