Evaluation of PCR, nested PCR, and fluorescent antibodies for detection of Giardia and Cryptosporidium species in wastewater

Mayer, Cynthia; Palmer, Carol

doi:10.1128/aem.62.6.2081-2085.1996

Cited by 117 publications

(34 citation statements)

References 24 publications

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“…Our results demonstrate the effectiveness of IMS in eliminating PCR inhibitors. Mayer and Palmer (1996) used IFA and nested PCR to evaluate the presence of cryptosporidial oocysts in sewage ef¯uent and reported a 70% agreement between the results from both methods. Chung et al (1998) showed there to be an 84% correlation between PCR and IFA in spiked municipal drinking water samples and a correlation of up to 97Á2% between nested PCR and IFA.…”

Section: Discussionmentioning

confidence: 99%

Detection of cryptosporidia and Cryptosporidium parvum oocysts in environmental water samples by immunomagnetic separation-polymerase chain reaction

Hallier-Soulier¹,

Guillot²

2000

J Appl Microbiol

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Cryptosporidium parvum has emerged as one of the most important new contaminants found in drinking water. Current protocols for the detection of cryptosporidia are time-consuming and rather inef®cient. We recently described an immunomagnetic separation±polymerase chain reaction (IMS±PCR) assay permitting highly sensitive detection of C. parvum oocysts in drinking water samples. In this study, a second IMS±PCR assay to detect all cryptosporidial oocysts was developed, and both IMS±PCR assays were optimized on river water samples. A comparative study of the two IMS±PCR assays and the classical detection method based on an immuno¯uorescence assay (IFA) was carried out on 50 environmental samples. Whatever the type of water sample, the discrepancy in C. parvum detection between the IFA and IMS±PCR took the form of IFA-negative/IMS±PCR-positive results, and was caused mainly by the greater sensitivity of IMS±PCR as compared with IFA. Of the 50 water samples, only ®ve tested positive for C. parvum using IMS±PCR, and could constitute a threat to human health. These results show that both IMS±PCR assays provide a rapid (1 d) and sensitive means of screening environmental water samples for the presence of cryptosporidia and C. parvum oocysts.

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Section: Discussionmentioning

confidence: 99%

Detection of cryptosporidia and Cryptosporidium parvum oocysts in environmental water samples by immunomagnetic separation-polymerase chain reaction

Hallier-Soulier¹,

Guillot²

2000

J Appl Microbiol

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“…Stinear et al (1996) developed a reverse transcription (RT)-peR based on the detection of mRNA from a C. parvum heat shock protein (hsp) that was capable of detecting a single viable oocyst from concentrated samples in a rapid (I-day) assay. Mayer and Palmer (1996) compared PCR, nested PCR, and fluorescent antibodies (IFA) for the detection of Cryptosporidium and Giardia in wastewater samples. They found a good correlation between the IFA and PCR detection methods and concluded that PCR may be a useful tool in the analysis of environmental samples for these organisms.…”

Section: Protozoamentioning

confidence: 99%

An Analytical Method for Trifluoroacetic Acid in Water and Air Samples Using Headspace Gas Chromatographic Determination of the Methyl Ester

Zehavi

Seiber

1996

Anal. Chem.

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An analytical method has been developed for the determination of trace levels of trifluoroacetic acid (TFA), an atmospheric breakdown product of several of the hydrofluorocarbon (HFC) and hydrochlorofluorocarbon (HCFC) replacements for the chlorofluorocarbon (CFC) refrigerants, in water and air. TFA is derivatized to the volatile methyl trifluoroacetate (MTFA) and determined by automated headspace gas chromatography (HSGC) with electron-capture detection or manual HSGC using GC/MS in the selected ion monitoring (SIM) mode. The method is based on the reaction of an aqueous sample containing TFA with dimethyl sulfate (DMS) in concentrated sulfuric acid in a sealed headspace vial under conditions favoring distribution of MTFA to the vapor phase. Water samples are prepared by evaporative concentration, during which TFA is retained as the anion, followed by extraction with diethyl ether of the acidified sample and then back-extraction of TFA (as the anion) in aqueous bicarbonate solution. The extraction step is required for samples with a relatively high background of other salts and organic materials. Air samples are collected in sodium bicarbonate-glycerin-coated glass denuder tubes and prepared by rinsing the denuder contents with water to form an aqueous sample for derivatization and analysis. Recoveries of TFA from spiked water, with and without evaporative concentration, and from spiked air were quantitative, with estimated detection limits of 10 ng/mL (unconcentrated) and 25 pg/mL (concentrated 250 mL:1 mL) for water and 1 ng/m(3) (72 h at 5 L/min) for air. Several environmental air, fogwater, rainwater, and surface water samples were successfully analyzed; many showed the presence of TFA.

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“…Molecular methods such as PCR have been proposed as an alternative to IFA for the detection of C. parvum in environmental samples (Mayer and Palmer 1996;Kaucner and Stinear 1998). The results of this study indicate that the cell wall antigenic components are more susceptible to microbial activity than the genome.…”

Section: Discussionmentioning

confidence: 88%

“…These results suggest that although the cell wall was digested, it protected the oocysts' DNA from extracellular nucleases. Mayer and Palmer (1996) compared the detection sensitivity of C. parvum oocysts from wastewater by IFA and PCR (Mayer and Palmer 1996). The presence of humic substances resulted in an inhibition of DNA synthesis by PCR.…”

Section: Discussionmentioning

confidence: 99%

Die-off of Cryptosporidium parvum in soil and wastewater effluents

Nasser¹,

Tweto²,

Nitzan³

2007

J Appl Microbiol

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Aims: To determine the effect of biotic and abiotic components of soil on the viability and infectivity of Cryptosporidium parvum, and evaluate the suitability of viability tests as a surrogate for oocyst infectivity under various environmental settings. Methods and Results: The die‐off of C. parvum in saturated and dry loamy soil was monitored over time by immunofluorescence assay (IFA) and PCR to estimate oocysts viability and by cell culture to estimate oocysts infectivity. Pseudomonas aeruginosa activity resulted in digestion of the outer layer of the oocysts, as demonstrated by loss of the ability to react in IFA. Whereas, P. aeruginosa activity did not affect the DNA amplification by PCR. A 1‐log reduction in the oocysts infectivity was observed at 30 °C in distilled water and in saturated soil while oocysts viability was unchanged. Incubation for 10 days in dry loamy soil at 32 °C resulted in a 3‐log10 reduction in their infectivity while no change of oocysts viability was recorded. Conclusions: Under low temperature, C. parvum oocysts may retain their infectivity for a long time. Soil desiccation and high temperatures enhance the die‐off rate of C. parvum. Significance and Impact of the Study: Previous die‐off studies of C. parvum used viability tests that do not necessarily reflect the oocyst infectivity. Under low temperatures, there was an agreement observed between viability and infectivity tests and oocysts retained their infectivity for a long time. Desiccation and high temperatures enhance the loss of infectivity of C. parvum. The presented die‐off data have significant implications on the management of wastewater reuse in warm environments.

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Evaluation of PCR, nested PCR, and fluorescent antibodies for detection of Giardia and Cryptosporidium species in wastewater

Cited by 117 publications

References 24 publications

Detection of cryptosporidia and Cryptosporidium parvum oocysts in environmental water samples by immunomagnetic separation-polymerase chain reaction

Detection of cryptosporidia and Cryptosporidium parvum oocysts in environmental water samples by immunomagnetic separation-polymerase chain reaction

An Analytical Method for Trifluoroacetic Acid in Water and Air Samples Using Headspace Gas Chromatographic Determination of the Methyl Ester

Die-off of Cryptosporidium parvum in soil and wastewater effluents

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