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Applied and Environmental Microbiology, September 2006, p. 5942-5947, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00927-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Lihua Xiao,1* Kerri A. Alderisio,2 and Jianlin Jiang1

Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30341,1 New York City Department of Environmental Protection, Valhalla, New York 105952

Received 19 April 2006/ Accepted 5 June 2006

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 microscopy-negative 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.

* Corresponding author. Mailing address: Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Building 22, Mail Stop F-12, 4770 Buford Highway, Atlanta, GA 30341-3717. Phone: (770) 488-4840. Fax: (770) 488-4454. E-mail: lxiao{at}cdc.gov.

Applied and Environmental Microbiology, September 2006, p. 5942-5947, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00927-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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