Immunomagnetic separation study applied to detection of Giardia spp. cysts and Cryptosporidium spp. oocysts in water samples

In this work we studied the IMS purification comparing acid (n = 12) and heat (n = 12) dissociation procedures and investigated the possible losses of target organism in this step. Reagent water samples were directly inoculated with Giardia spp. cysts and Cryptosporidium spp. oocysts (BTF Easy Seed™). Acid dissociation showed higher mean recovery efficiency and precision than heat dissociation for Giardia spp. cysts and Cryptosporidium spp. oocysts, but there were not significant statistic differences for Cryptosporidium spp. oocysts. Mean recovery efficiency for both protozoa were in accordance with the acceptance criteria of Method 1623 by acid and heat dissociation. The bead–cyst/bead–oocysts dissociation procedure is fundamental for better results whereas a significant loss of organisms occurs in this step.

Effective Concentration and Detection of Cryptosporidium, Giardia, and the Microsporidia from Environmental Matrices

Cryptosporidium spp., Giardia spp., and members of Microsporidia are enteropathogenic parasites of humans and animals, producing asymptomatic to severe intestinal infections. To circumvent various impediments associated with current detection methods, we tested a method providing multistage purification and separation in a single, confined step. Standard real-time PCR was used as a detection method. Samples spiked with C. parvum and G. intestinalis were split for comparison to standard Method 1623. Results were equivalent to immunomagnetic procedures for Cryptosporidium, and Giardia. Overall percent recovery for Cryptosporidium with Method 1623 averaged 26.89% (std 21.44%; min = 0%; max = 73%) and was similar but less variable for qPCR method at an estimated average of 27.67 (std 17.65%; min = 5%; max = 63%). For Giardia, Method 1623 had an overall average recovery of 27.11% (std 17.98%; min = 1%; max = 58%), while multistage purification and qPCR had an estimated lower overall recovery at 18.58% (std 13.95%; min = 0%; max = 35%). Microsporidia were also readily detected with an estimated recovery of 46.81% overall (std 17.66%; min = 18%; max = 70%) for E. intestinalis and 38.90% (std 14.36%; min = 13%; max = 62%) for E. bieneusi.

Occurrence, Source, and Human Infection Potential of Cryptosporidium and Giardia spp. in Source and Tap Water in Shanghai, China

ABSTRACTGenotyping studies on the source and human infection potential ofCryptosporidiumoocysts 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 forCryptosporidiumand 18% forGiardiaby Method 1623, whereas 22% were positive forCryptosporidiumand 10% forGiardiaby microscopy of CCF concentrates. When CCF was combined with PCR for detection, the occurrence ofCryptosporidium(28%) was similar to that obtained by Method 1623. Genotyping ofCryptosporidiumin 17 water samples identified the presence ofC. andersoniin 14 water samples,C. suisin 7 water samples,C. baileyiin 2 water samples,C. meleagridisin 1 water sample, andC. hominisin 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.Cryptosporidiumoocysts 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 ofCryptosporidiumcontamination in water, although the results from this method are semiquantitative.

Identification of Particle Size Classes Inhibiting Protozoan Recovery from Surface Water Samples via U.S. Environmental Protection Agency Method 1623

ABSTRACT Giardia species recovery by U.S. Environmental Protection Agency method 1623 appears significantly impacted by a wide size range (2 to 30 μm) of particles in water and organic matter. Cryptospori dium species recovery seems negatively correlated only with smaller (2 to 10 μm), presumably inorganic particles. Results suggest constituents and mechanisms interfering with method performance may differ by protozoan type.