Application of Cation-Coated Filter Method to Detection of Noroviruses, Enteroviruses, Adenoviruses, and Torque Teno Viruses in the Tamagawa River in Japan

The occurrence of human enteric viruses in surface water in the Tamagawa River, Japan, was surveyed for 1 year, from April 2003 to March 2004. Sixty-four samples were collected from six sites along the river, and 500 ml of the sample was concentrated using the cation-coated filter method, which was developed in our previous study. This method showed recovery yields of 56% ± 32% (n = 37) for surface water samples inoculated with polioviruses. More than one kind of tested virus was detected in 43 (67%) of 64 samples by TaqMan PCR. Noroviruses and adenoviruses were detected in a high positive ratio; 34 (53%), 28 (44%), and 29 (45%) of 64 samples were positive for norovirus genotype 1 and genotype 2 and adenoviruses, respectively. The mean concentrations of norovirus genotype 1 or genotype 2 determined by real-time PCR were 0.087 and 0.61 genome/ml, respectively, showing much higher values in winter (0.21 genome/ml for genotype 1 and 2.3 genomes/ml for genotype 2). Enteroviruses were detected by both direct PCR (6 of 64 samples; 9%) and cell culture PCR (2 of 64 samples; 3%). Torque teno viruses, emerging hepatitis viruses, were also isolated in three samples (5%). The concentration of total coliforms and the presence of F-specific phages showed a high correlation with the presence of viruses, which suggested that the simultaneous use of total coliforms and F-specific phages as indicators of surface water may work to monitor viral contamination.     

Application of cation-coated filter method to detection of noroviruses, enteroviruses, adenoviruses, and torque teno viruses in the Tamagawa River in Japan

The occurrence of human enteric viruses in surface water in the Tamagawa River, Japan, was surveyed for 1 year, from April 2003 to March 2004. Sixty-four samples were collected from six sites along the river, and 500 ml of the sample was concentrated using the cation-coated filter method, which was developed in our previous study. This method showed recovery yields of 56% +/- 32% (n = 37) for surface water samples inoculated with polioviruses. More than one kind of tested virus was detected in 43 (67%) of 64 samples by TaqMan PCR. Noroviruses and adenoviruses were detected in a high positive ratio; 34 (53%), 28 (44%), and 29 (45%) of 64 samples were positive for norovirus genotype 1 and genotype 2 and adenoviruses, respectively. The mean concentrations of norovirus genotype 1 or genotype 2 determined by real-time PCR were 0.087 and 0.61 genome/ml, respectively, showing much higher values in winter (0.21 genome/ml for genotype 1 and 2.3 genomes/ml for genotype 2). Enteroviruses were detected by both direct PCR (6 of 64 samples; 9%) and cell culture PCR (2 of 64 samples; 3%). Torque teno viruses, emerging hepatitis viruses, were also isolated in three samples (5%). The concentration of total coliforms and the presence of F-specific phages showed a high correlation with the presence of viruses, which suggested that the simultaneous use of total coliforms and F-specific phages as indicators of surface water may work to monitor viral contamination.     

Rapid detection of Salmonella spp. in food by use of the ISO-GRID hydrophobic grid membrane filter.

A rapid hydrophobic grid-membrane filter (HGMF) method was developed and compared with the Health Protection Branch cultural method for the detection of Salmonella spp. in 798 spiked samples and 265 naturally contaminated samples of food. With the HGMF method, Salmonella spp. were isolated from 618 of the spiked samples and 190 of the naturally contaminated samples. The conventional method recovered Salmonella spp. from 622 spiked samples and 204 unspiked samples. The isolation rates from Salmonella-positive samples for the two methods were not significantly different (94.6% overall for the HGMF method and 96.7% for the conventional approach), but the HGMF results were available in only 2 to 3 days after sample receipt compared with 3 to 4 days by the conventional method.     

Determination of raloxifene and its glucuronides in human urine by liquid chromatography-tandem mass spectrometry assay

A selective, sensitive, accurate and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of raloxifene and its three glucuronides: raloxifene-6-β-glucuronide (M1), raloxifene-4'-β-glucuronide (M2), raloxifene-6,4'-diglucuronide (M3) in urine samples is presented in this paper. To our knowledge the developed analytical method is the first fully validated method capable of simultaneous determination of raloxifene and its glucuronides in real urine samples. Moreover, for the first time a method for determination of raloxifene diglucuronide in relevant biological samples was introduced. Metabolites were obtained by a bioconversion process of raloxifene to its glucuronides using the microorganism Streptomyces sp. and were used as standards for validation. Urine samples were introduced to a simple solid phase extraction prior to the analysis by LC-MS/MS. The method was linear in a wide range with high determination coefficient (r(2) > 0.997). The limits of quantification achieved were 1.01, 1.95, 2.83 and 4.69 nM for raloxifene, M1, M2 and M3, respectively. The recoveries were higher than 92.5%, the accuracy was within 100 ± 8.8% and the precision was better than 12% for all compounds. The developed method was successfully applied to the real urine samples and showed to be appropriate for use in further research of still not completely discovered raloxifene pharmacokinetics. Furthermore, the presented method could also serve for a potential application in anti-doping analysis.     

Blend uniformity analysis using stream sampling and near infrared spectroscopy

A near infrared spectroscopic method was developed to determine drug content in a 20% (wt/wt) ibuprofen and spray-dried hydous lactose blend. A blending profile was obtained after blending for 0.5, 1, 3, 5, 10, and 20 minutes. Stream sampling was used to collect about 20 blend samples at each of the blending times from a laboratory scale V-blender. The samples collected were used to develop a near infrared calibration model. The calibration model was then used to determine the drug content of unknown samples from 2 validation blends. The validation blends were not included in the calibration model; they were used to evaluate the effectiveness of the calibration model. A total of 45 samples from the 2 validation blends were predicted by the near infrared calibration model and then analyzed by a validated UV spectrophotometric method. The root mean square error of prediction for the first validation blend was 5.69 mg/g and 3.30 mg/g for the samples from the second blend. A paired t test at the 95% confidence level did not indicate any differences between the drug content predicted by the near infrared spectroscopy (NIRS) method and the validated UV method for the 2 blends. The results show that the NIRS method could be developed while the blending profile is generated and used to thoroughly characterize a new formulation during development by analyzing a large number of samples. The new formulation could be transferred to a manufacturing plant with an NIRS method to facilitate blend uniformity analysis.     

Comparison of LST + MUG broth technique and conventional method for the enumeration of Escherichia coli in foods

AIMS: To reduce the analysis time needed for the enumeration of Escherichia coli, a rapid fluorogenic method (MUG) which takes only 48 h was compared with the standard most probable number (MPN) method which takes 6 days as described in the International Standards Organization (ISO). This study provides reliability data for the fluorogenic method applied to certain foods. METHODS AND RESULTS: Both methods were applied to 500 food samples which were analysed for E. coli enumeration. Agreement between the two methods was found in 409 (81 x 8%) samples; 81 (16 x 2%) samples gave higher values by the fluorogenic method, and only 10 (2 x 0%) samples were more effectively assayed by the ISO method. According to statistical analysis, the reliability between the methods was r = 0 x 9706, r(2) = 0 x 9421 and Cronbach's alpha = 0 x 9851. While all three values showed a high degree of correlation (P < 0 x 0001) between the two methods, McNemar's test demonstrated a significant difference between them, indicating that the MUG method was more reliable than the ISO method. CONCLUSIONS: The data suggest that the fluorogenic method is more reliable and shorter to perform than the standard ISO method. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison of the two methods may provide a rapid and more reliable alternative for the enumeration of E. coli in food samples.     

大花红天素分离、合成及其含量分析

研究大花红天素的分离、合成,并对大花红天素的含量进行分析测定。通过东京化成HP-20和硅胶柱色谱进行分离;以2-甲基-3-丁烯-2-醇和溴代四乙酰葡萄糖通过SN2反应合成大花红天素,合成收率为18％;通过HPLC-ELSD对大花红天素的含量进行分析,通过对8种样品的分析,测定大花红景天和红景天饮片中分别含大花红天素2．06％、0.83％,其他样品中未检出大花红天素。作者对大花红天素进行了首次合成并建立了相应的分析测定方法。     

Dot-ELISA法检测血液及唾液HIV抗体的应用评价

评价人类免疫缺陷病毒1+2型抗体检测试剂盒(Dot-ELISA法)检测血清和唾液样本的临床性能。采用对照试验研究,选取背景清晰的研究对象200例,采集同一研究对象的血清和唾液样本,应用万泰生物药业公司生产的人类免疫缺陷病毒1+2型抗体检测试剂盒作为考核试剂,法国生物梅里埃公司生产的人类免疫缺陷病毒抗体诊断试剂盒(ELISA法)作为参考试剂,考核试剂检测结果与参考试剂及研究对象背景进行比较分析。考核试剂检测血清HIV抗体与参考试剂相比较,阳性符合率100%,阴性符合率100%,总符合率100%,Kappa值1.00,一致性为最强;考核试剂检测唾液HIV抗体与参考试剂检测结果相比较,阳性符合率98.78%,阴性符合率100%,总符合率99.50%,Kappa值0.99,一致性为最强。Dot-ELISA法人类免疫缺陷病毒1+2型抗体检测试剂盒对血清及唾液样本检测性能优越,适合HIV抗体快速筛查。     

Genetic variability of hepatitis B virus isolates among population of Shuryskarsky area of Yamal-Nenets autonomous region

The purpose of this work was to determine occurrence of serological markers of hepatites B and to describe subtypes of a superficial antigen and genotypes of hepatitis B virus (HBV) isolates among indigenous population of Yamal-Nenets Autonomous Region (YNAR), Russia. METHODS: We investigated 657 serum samples from inhabitants of Shuryskarsky area of YNAR. ELISA method was used to define the hepatitis B markers: HBsAg, anti-HBs (total) and anti-HBc (IgG and IgM). The HBsAg-positive samples were PCR-tested for the presence of HBV DNA. Genotyping of isolates was by sequencing of the Pre-Sl/Pre-82/S region of HBV genome and phylogenetic analysis. Definition of HBsAg subtypes was executed by two methods: ELISA with subtype-specific monoclonal antibodies and S-gene nucleotide sequence analysis. RESULTS: The following occurrence of hepatitis B markers was observed: HBsAg - 3.2%, anti-HBs (total) - 36.2%, anti-HBc IgG - 30.3%, anti-HBc IgM - 1.6%. Frequency of carrying even one of the markers in the observed population was 47.5%. HBV DNA was found in 17 HBsAg-positive samples. Pre-SI, Pre-S2 and S regions sequences were determined for all HBV DNA-positive samples. The phylogenetic analysis showed an accessory of all investigated HBV isolates to genotype D. HBsAg subtypes distribution appeared the following: ayw2 - 23.5%, ayw3 - 70.6%, adw2 - 5.9%. Results of definition of the subtype ELISA method and by the analysis of S gene nucleotide sequences have coincided in 10/11 (90.1%) cases. CONCLUSIONS: The indigenous population of Shuryskarsky area of YNAR belongs to groups with average HBV carrying. Absolute domination of genotype D (subtypes ayw2, ayw3 and adw2) was revealed. High percentage of concurrence of HBsAg subtypes detected by the ELISA method and method of the analysis of S gene primary structure (90%) was observed. Sequencing of HBV S-gene is preferable to define HBsAg subtypes.     

Evaluation of two automated enzyme-immunoassays for detection of thermophilic campylobacters in faecal samples from cattle and swine.

We evaluated the performance of two enzyme-immunoassays (EIA) for the detection of naturally occurring, thermophilic Campylobacter spp. found in faecal samples from cattle (n = 21 and n = 26) and swine (n = 43) relative to the standard culture method, and also assuming that none of the tests was the definitive standard. The primary isolation both for the culture and the EIA methods was carried out by overnight selective enrichment in Preston broth. The results showed good sensitivities for both EIA methods in cattle (95% and 84%) and swine (88% and 69%) samples. However, when testing cattle samples, EIA-2 method resulted in a rather low specificity (32%). This seemed to be partially due to the isolation of nonthermophilic species. In conclusion, EIA-1 method may provide a simple and fast tool with good accuracy in cattle and swine samples for automated screening of large number of samples.     

Detection of noroviruses in tap water in Japan by means of a new method for concentrating enteric viruses in large volumes of freshwater

A virus concentration method using a cation-coated filter was developed for large-volume freshwater applications. Poliovirus type 1 (LSc 2ab Sabin strain) inoculated into 40 ml of MilliQ (ultrapure) water was adsorbed effectively to a negatively charged filter (Millipore HA, 0.45- micro m pore size) coated with aluminum ions, 99% (range, 81 to 114%) of which were recovered by elution with 1.0 mM NaOH (pH 10.8) following an acid rinse with 0.5 mM H(2)SO(4) (pH 3.0). More than 80% poliovirus recovery yields were obtained from 500-ml, 1,000-ml, and 10-liter MilliQ water samples and from tap water samples. This method, followed by TaqMan PCR detection, was applied to determine the presence of noroviruses in tap water in Tokyo, Japan. In a 14-month survey, 4 (4.1%) and 7 (7.1%) of 98 tap water samples (100 to 532 liters) contained a detectable amount of noroviruses of genotype 1 and genotype 2, respectively. This method was proved to be useful for surveying the occurrence of enteric viruses, including noroviruses, in large volumes of freshwater.     

Determination of lycopene in tissues and plasma of rats by normal-phase high-performance liquid chromatography with photometric detection

An analytical method for the determination of lycopene in tissues and plasma of rats is described. The method was validated for the determination of lycopene in liver and plasma with respect to selectivity, linearity, accuracy, recovery and precision. Following precipitation of proteins with water–ethanol plasma was extracted with hexane; tissues were extracted with acetone followed by precipitation of proteins with water–ethanol and extraction of lycopene with hexane. Separation and quantification of geometrical isomers of lycopene was achieved by normal-phase HPLC with UV/VIS detection at 471 nm. The method proved to be selective and specific for lycopene in plasma and liver. Detector response was linear in the range from 2 ng/g to 10 μg/g liver and 0.5 ng/ml to 2 μg/ml plasma, respectively. Average recoveries ranged from 96 to 101% in spiked liver samples and from 91 to 94% in spiked plasma samples. Intra-day variability (C.V.) was ≤6% and ≤5% in liver and plasma, respectively. Inter-day precision was ≤9% for liver samples and ≤6% for plasma samples. The procedures were successfully applied to the sample analysis of pharmacokinetic and metabolism studies.     

Enrichment followed by quantitative PCR both for rapid detection and as a tool for quantitative risk assessment of food-borne thermotolerant campylobacters

As part of a large international project for standardization of PCR (Food-PCR; www.pcr.dk), a multiplex, multiplatform, ready-to-go enrichment followed by a real-time PCR method, including an internal amplification control, was developed for detection of food-borne thermotolerant campylobacters in chickens. Chicken rinse samples were enriched in Bolton broth for 20 h, a simple and rapid (1-h) resin-based DNA extraction was performed, and DNA samples were then tested with two instrument platforms: ABI-PRISM 7700 and RotorGene 3000. The method was validated against an International Standard Organization (ISO)-based culture method by testing low, medium, and high levels of 12 spiked and 66 unspiked, presumably naturally contaminated, chicken rinse samples. In the RotorGene, a positive PCR response was detected in 40 samples of the 66. This was in complete agreement with the enriched ISO culture. The ABI-PRISM 7700 missed one culture-positive sample. Positive samples contained 10(2) to 10(7) CFU/ml after enrichment in Bolton broth. In the enriched samples a detection probability of 95% was obtained at levels of 1 x 10(3) and 2 x 10(3) CFU/ml in the RotorGene and ABI-PRISM, respectively. The amplification efficiency in both platforms was 90%, although the linear range of amplification of purified genomic DNA was 1.5 x 10(1) to 1 x 10(7) (R(2) = 1.00) for the RotorGene and 10(3) to 10(7) (R(2) = 0.99) for the ABI-PRISM. In RotorGene and ABI-PRISM the levels of precision of detection as determined by standard deviation (coefficients of variation) of 6-carboxyfluorescein (FAM) threshold cycle (Ct) values were 0.184 to 0.417 (0.65 to 2.57%) and 0.119 to 0.421 (0.59 to 1.82%), respectively. The results showed a correlation (R(2)) of 0.94 between the target FAM Ct values and CFU per milliliter of enriched naturally contaminated chicken samples, which indicates PCR's additional potential as a tool for quantitative risk assessment. Signal from the internal amplification control was detected in all culture-negative samples (VIC Ct: 23.1 to 28.1). The method will be taken further and validated in an international collaborative trial with regard to standardization.     

A rapid method for extracting oocyst DNA from Cryptosporidium-positive human faeces for outbreak investigations

We describe a rapid method for extracting and concentrating Cryptosporidium oocysts from human faecal samples with subsequent DNA preparation for mainstream PCR applications. This method consists of extracting faecal lipids using a modified water-ether treatment and releasing DNA from semi-purified oocysts by freeze thawing in lysis buffer. Following immunomagnetisable separation (IMS), recovery rates of 29.5%, 43.2% and 49.8% were obtained from oocyst-negative solid, semi-solid and liquid faeces, respectively, seeded with 100 +/- 2 C. parvum oocysts, which were enumerated by flow cytometry. A retrospective analysis was conducted on 92 positive human faecal samples including 78 oocyst-positive cases from 2 UK cryptosporidiosis outbreaks (outbreak A = 34 samples, outbreak B = 44 samples) and 14 oocyst-positive, sporadic cases. We used primers targeting the Cryptosporidium oocyst wall protein gene (COWP; STN-COWP), the 18S rRNA (direct PCR) and the dihydrofolate reductase gene (dhfr, MAS-PCR) fragments to evaluate extracted DNA by PCR. PCR inhibitors were present in 20 samples when template was co-amplified with the 18S rRNA gene primers and an internal control. Template dilution (1/5) in polyvinylpyrrolidone (10 mg ml(-1), pH 8.0) transformed four PCR-negative samples to PCR-positive and increased amplicon intensity in previously positive samples. Eighteen of 20 PCR-negative samples produced visible amplicons when Taq polymerase concentration in the STN-COWP PCR was increased from 2.5 to 5 U. The STN-COWP PCR assay amplified 90 of 92 samples (97.8%) and the MAS-PCR assay amplified 70 of 92 samples (76.1%) tested. In the absence of inhibitors, DNA equivalent to 3 C. parvum oocysts was amplified.     

A novel method and simple apparatus for the detection of thermophilic Campylobacter spp. in chicken meat products

Conventional procedures for isolation of thermophilic Campylobacter spp. from chicken are complex, labor intensive, and time-consuming. The objective of this study was to create a novel Campylobacter culturing apparatus. A main concept of the device was based on the ability of Campylobacter to pass through a 0.45 microm pore size filter in viscous media. Preliminary study demonstrated that only viable Campylobacter moved through the membrane filter and could multiply in the enrichment culture. C. jejuni, C. coli, C. lari, and C. upsaliensis in the chicken samples were detected at cell concentrations as low as 10 cfu/g, after 24 h incubation at 42 degrees C. In total, 84 retail chicken samples were comparatively studied using both conventional method and apparatus. Sixteen samples (19.05%) were positive by the apparatus method; 14 (16.66%) of these positive samples contained C. coli and 2 (2.38%) contained C. jejuni. With the conventional method, 7 (8.33%) samples were positive 7 (8.33%) with C. coli. In conclusion, the apparatus detected more positive samples than did the conventional culture method.     

Determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in human urine

A method for simultaneous determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in urine is described. These compounds are metabolites of N-methyl-2-pyrrolidone, a powerful and widely used organic solvent. 5-Hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide were purified from urine by adsorption to a C₈ solid-phase extraction column and then elution by ethyl acetate–methanol (80:20). After evaporation, the samples were derivatised at 100°C for 1 h by bis(trimethylsilyl)trifluoroacetamide. Ethyl acetate was then added and the samples were analysed by gas chromatography–mass spectrometry in the electron impact mode. The extraction recovery for 5-hydroxy-N-methylpyrrolidone was about 80% while that for 2-hydroxy-N-methylsuccinimide was about 30%. The intra-day precision for 5-hydroxy-N-methylpyrrolidone was 2–4% and the between-day precision 4–21% (4 and 60 μg/ml). The intra-day precision for 2-hydroxy-N-methylsuccinimide was 4–8% and the between-day precision 6–7% (2 and 20 μg/ml). The detection limit was 0.2 μg/ml urine for both compounds. The method is applicable for analysis of urine samples from workers exposed to N-methyl-2-pyrrolidone.     

Analysis of T-2 and HT-2 toxins in oats and other cereals by means of HPLC with fluorescence detection

Bearing in mind the high toxicity of T-2 and HT-2 toxins which occur in cereals (mainly in oats) EU plans legal limits for these mycotoxins. The occurrence data are insufficient because reliable and sensitive analysis methods are not available. A sensitive HPLC gradient method was developed which is applicable with common HPLC equipment (HPLC with fluorescence detection). After extraction of the toxins from sample matrix with methanol/water the diluted extracts were cleaned-up using immunoaffinity columns and then derivatized with 1-anthroylnitrile/DMAP. The T-2 and HT-2 toxins were separated from peaks of the cereal matrix and derivatization reagent by means of a relatively complex HPLC gradient method. The method was validated for oats, wheat, rye, barley, and maize. The recovery rates were in the range of 70–99%, the precision (RSD_R) of 3–8%. The limits of detection of T-2 and HT-2 toxins were 1 μg/kg. A total of 119 samples of cereals and cereal products was analyzed according to the optimized method. The analyses of 54 samples of dehulled oats and of 11 samples of processed oat products from food industry had a contamination frequency of 100%. The contents (sum of T-2 and HT-2 toxins) amounted to 3 to 174 μg/kg for the dehulled oats and to 4 to 48 μg/kg for the processed oat products. 29 samples of maize and maize products had a contamination frequency of 80% (2–106 μg/kg in the sum of T-2 and HT-2 toxins). In the samples of wheat and barley the toxins were detected only occasionally (contents: 1–10 μg/kg), in rye not at all.     

改良离心集菌法检测痰内抗酸杆菌的研究

目的研究改良离心集菌法,测定其是否能使抗酸杆菌的诊断更为安全简便。方法选取传统的抗酸杆菌直接涂片法1＋和1＋以下的阳性标本100例,用改良离心集菌法复检;再配制浓度为10000、5000、1000和100条／mL的痰菌液各100份,分别用直接涂片法和改良离心集菌法检测其检出限和检出率;最后以日常门诊标本1036例分别用两种方法进行临床应用检测和分析。结果100例直接涂片法1＋和1＋以下的阳性标本改为改良离心集菌法复检,结果为4＋有93例、3＋为7例;直接涂片法和改良集菌法的检出限分别为1000和100条／mL;10000、5000、1000和100条／mL的痰菌液用直接涂片法和改良离心集菌法检测其检出率分别为97％、53％、5％、0％和100％、100％、94％、7％。1036例日常标本用直接涂片法和改良集菌法检测的阳性率分别12．74％和16．47％。结论改良离心集菌法的阳性率高于直接涂片法,检出限低于直接涂片法,不但大大改善了实验室和实验人员生物安全,还弥补了直接涂片法中对结核杆菌在痰液中分布不均一性带来的漏检的方法局限性,因此改良离心集菌法是安全、简便的抗酸杆菌实验室值得推广使用的新诊断方法。     

Occurrence, source, and human infection potential of cryptosporidium and Giardia spp. in source and tap water in shanghai, china

Genotyping studies on the source and human infection potential of Cryptosporidium oocysts in water have been almost exclusively conducted in industrialized nations. In this study, 50 source water samples and 30 tap water samples were collected in Shanghai, China, and analyzed by the U.S. Environmental Protection Agency (EPA) Method 1623. To find a cost-effective method to replace the filtration procedure, the water samples were also concentrated by calcium carbonate flocculation (CCF). Of the 50 source water samples, 32% were positive for Cryptosporidium and 18% for Giardia by Method 1623, whereas 22% were positive for Cryptosporidium and 10% for Giardia by microscopy of CCF concentrates. When CCF was combined with PCR for detection, the occurrence of Cryptosporidium (28%) was similar to that obtained by Method 1623. Genotyping of Cryptosporidium in 17 water samples identified the presence of C. andersoni in 14 water samples, C. suis in 7 water samples, C. baileyi in 2 water samples, C. meleagridis in 1 water sample, and C. hominis in 1 water sample. Therefore, farm animals, especially cattle and pigs, were the major sources of water contamination in Shanghai source water, and most oocysts found in source water in the area were not infectious to humans. Cryptosporidium oocysts were found in 2 of 30 tap water samples. The combined use of CCF for concentration and PCR for detection and genotyping provides a less expensive alternative to filtration and fluorescence microscopy for accurate assessment of Cryptosporidium contamination in water, although the results from this method are semiquantitative.