Detection of Cryptosporidium oocysts in water: effect of the number of samples and analytic replicates on test results

Abstract

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.

FIG. 1.

PCR amplification of DNA from samples and subsamples of storm water collected from the same N5 basin site on 27 October 2003. DNA 8509, 8510, 8511, 8512, 8513, 8514, 8515, and 8516 are from samples and subsamples 1120, 1121, 1122A, 1122B, 1122C, 1122D, 1123A, and 1123B, respectively. Lanes 1 to 14 are PCR products using 0.5 μl of DNA suspension, lanes 15 to 28 are PCR products using 1 μl of DNA suspension, lanes 29 to 42 are PCR products using 1.5 μl of DNA suspension, and P is the positive control (Cryptosporidium serpentis DNA). DNA labeled “a” and “b” are from two different DNA extractions using 0.5 ml of packed pellet from the same subsample. Eight, 9, and 11 of the 14 DNA preparations were amplified when 0.5, 1, and 1.5 μl of DNA suspension, respectively, was used in the PCR.

FIG. 2.

Diversity of Cryptosporidium genotypes in storm water samples as indicated by RFLP analysis (one PCR run of one batch of samples taken on 19 September 2003). DNA 8218, 8219, and 8220 are from three subsamples of one sample; 8221 and 8222 are from two subsamples of a second sample; and 8223 and 8224 are from two subsamples of the third sample (a, b, and c are different DNA extractions of the same subsample). Most PCR products had only one subtype (except for lane 7), even though most samples or subsamples had more than one genotype. Upper panel, SspI digestion products; lower panel, VspI digestion products; P, positive control (C. serpentis).