Improvement of recoveries for the determination of protozoa Cryptosporidium and Giardia in water using method 1623

Hu, Jiangyong; Feng, Yaoyu; Ong, Say Leong; Ng, Wun Jern; Song, Lianfa; Tan, Xin; Chu, Xiaona

doi:10.1016/j.mimet.2004.04.013

Cited by 52 publications

(31 citation statements)

References 5 publications

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“…The cartridge/ foam filter and immunomagnetic separation, for their quickly filtration and specificity, were recommended for Cryptosporidium detection by the method 1623 of the USEPA, but their high cost makes it difficult to conduct environmental survey in large scale. Meanwhile, many studies have noted declines in Cryptosporidium and Giardia recovery levels to near or below minimum acceptable levels in waters of high turbidity or high total suspended solid (Krometis et al 2009;Feng et al 2003;Hu et al 2004). It is possible that Cryptosporidium oocysts, which could be adhered to particles, were still retained in the filter after elution process (Feng et al 2003).…”

Section: Initial Precision and Recovery Of Cryptosporidium Parvum Oocmentioning

confidence: 99%

Occurrences and genotypes of Cryptosporidium oocysts in river network of southern-eastern China

Xiao

Chen

et al. 2011

Parasitol Res

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Transportation of Cryptosporidium oocysts in river type source water is of great concern in an area where extensive human activities exist. In this study, a total of 47 samples were collected from Tongxiang, China, where drinking source water was taken from a complicated river network system, by three sampling campaigns over a rainy season in 2009, to reveal the presence, genotypes, and likely source of Cryptosporidium oocysts in river water. Immunofluorescence microscopy analyses show that 37 (78.7%) were Cryptosporidium positive, with a mean concentration of 0.51 oocysts per liter. These results suggest that the protozoa were commonly distributed in the river network type source water of Tongxiang with a relatively low concentration level. PCR analysis was used to determine the species/genotypes of Cryptosporidium, which revealed the presence of the animal related species/ genotypes including Cryptosporidium suis, Cryptosporidium fragile, and the avian III, pig II, cervine genotypes. Three of them were also detected in wastewater samples taken from neighboring animal farms, showing that farm animals rather than human might be the major pollution sources. This is the first report on simultaneous detection and genotyping of Cryptosporidium oocysts from surface water in China.

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Section: Initial Precision and Recovery Of Cryptosporidium Parvum Oocmentioning

confidence: 99%

Occurrences and genotypes of Cryptosporidium oocysts in river network of southern-eastern China

Xiao

Chen

et al. 2011

Parasitol Res

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“…This largely involves the concentration of oocysts by filtration, isolation of oocysts by immunomagnetic separation (IMS), staining of recovered oocysts with a fluorescent antibody and 4Ј,6-diamidino-2-phenylindole dihydrochloride (DAPI), and microscopic detection and enumeration of the stained oocysts (20). The method has been evaluated extensively with water samples seeded with known numbers of oocysts (2,3,6,7,14,16,18,22). Nevertheless, the method suffers from variations in oocyst recovery rates and an inability to differentiate species (23).…”

mentioning

confidence: 99%

Detection of Cryptosporidium Oocysts in Water: Effect of the Number of Samples and Analytic Replicates on Test Results

Xiao

Alderisio²,

Jiang

2006

Appl Environ Microbiol

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Due to the small number of Cryptosporidium oocysts in water, the number of samples taken and the analyses performed can affect the results of detection. In this study, 42 water samples were collected from one watershed during 20 storm events over 1 year, including duplicate or quadruplicate samples from 16 storm events. Ten samples from four events had three to eight subsamples. They were processed by EPA method 1623, and Cryptosporidium oocysts present were detected by immunofluorescent microscopy or PCR. Altogether, 24 of 39 samples (47 of 67 samples and subsamples) analyzed by microscopy were positive for Cryptosporidium. In contrast, 36 of 42 samples (62 of 76 samples and subsamples) were positive by PCR, including 10 microscopy-negative samples (13 microscopynegative samples and subsamples). Six of the 24 microscopy-positive samples were negative by PCR, and all samples had one or less oocyst in a 0.5-ml packed pellet volume calculated. Discordant results were obtained by microscopy and PCR from six and three of the storm events, respectively, with multiple samples. Discordant microscopy or PCR results were also obtained among subsamples. Most of the 14 Cryptosporidium genotypes were found over a brief period. Cryptosporidium-positive samples had a mean of 1.9 genotypes per sample, with 39 of the 62 positive samples/subsamples having more than one genotype. Samples/subsamples with more than one genotype had an overall PCR-positive rate of 73%, compared to 34% for those with one genotype. The PCR amplification rate of samples was affected by the volume of DNA used in PCR.

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“…Most noted a decrease in the recovery level of C. parvum oocysts after filtration (Feng et al 2003;Hu et al 2004). Our preliminary studies allowed us to determine that the extraction of DNA from C. parvum oocysts is most effective when it is preceded with cycles of liquid nitrogen/water baths incubation and with the use of lysate buffer and overnight proteinase K digestion (Adamska et al 2011).…”

Section: Discussionmentioning

confidence: 99%

“…Although only ten years have passed since the United States Environmental Protection Agency (USEPA) introduced the 1622 and 1623 methods (Anonymous, 2005a,b), these are in need of improvement (Hu et al 2004;Skotarczak 2009Skotarczak , 2010Adamska et al 2010). In both methods, the filtration of water samples of 10-50 l volume is carried out with the use of several kinds of filters in accordance with the producer's recommendations.…”

Section: Discussionmentioning

confidence: 99%

Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR

Adamska¹,

Leońska-Duniec²,

Sawczuk³

et al. 2012

Vet. Med.

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ABSTRACT:Cryptosporidium parvum is a common intestinal protozoan parasite infecting humans and a wide range of animals, whose diagnostics present considerable difficulties. These arise from the exceptionally robust nature of the oocyst's walls, which necessitates more stringent treatments for disruption and recovery of DNA for analysis using molecular methods. In the case of water, which is the major source of Cryptosporidium oocysts, investigations concern the detection of the presence of the oocysts. Their concentration in water is very low, and moreover, many substances that may have significance as inhibitors of DNA amplification, are present in environmental water and stool. We have carried out trials in order to assess the effectiveness of recovery of C. parvum oocysts, from spiked environmental and distilled water samples, filtrated and concentrated with the use of special laboratory equipment. Inactivation of inhibitors was carried out with use of bovine serum albumin (BSA) in PCR mixes at ten different concentrations. DNA extraction was carried out from stool samples spiked with C. parvum oocysts, concentrated using two methods, and unconcentrated. Nested PCR and a TaqMan nested real time PCR assay, targeting the 18S rRNA gene, was used to detect C. parvum DNA in spiked water and additionally in spiked stool samples. The obtained results showed that losses of C. parvum oocysts occur during the filtration and concentration of spiked water samples. The addition of small amounts of BSA (5-20 ng/µl) to PCR and TaqMan PCR mixes increases the sensitivity of both methods, but a high concentration of BSA (100 ng/µl and above) has an inhibiting effect on the polymerase reaction. The extraction of DNA from C. parvum oocysts from spiked stool samples preceded by concentration with PBS, ether and Percoll resulted in a higher copy number of the 18S rRNA gene.

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Improvement of recoveries for the determination of protozoa Cryptosporidium and Giardia in water using method 1623

Cited by 52 publications

References 5 publications

Occurrences and genotypes of Cryptosporidium oocysts in river network of southern-eastern China

Occurrences and genotypes of Cryptosporidium oocysts in river network of southern-eastern China

Detection of Cryptosporidium Oocysts in Water: Effect of the Number of Samples and Analytic Replicates on Test Results

Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR

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