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

2015 ◽  
Vol 16 (1) ◽  
pp. 144-149 ◽  
Author(s):  
Diego de Oliveira Pinto ◽  
Luciana Urbano ◽  
Romeu Cantusio Neto
Keyword(s):  
Water Samples ◽  
Immunomagnetic Separation ◽  
Significant Loss ◽  
Acid Dissociation ◽  
Recovery Efficiency ◽  
Acceptance Criteria ◽  
Cryptosporidium Spp ◽  
Method 1623

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.

Immunomagnetic separation of Cryptosporidium oocysts from water samples: round robin comparison of techniques

1997 ◽  
Vol 35 (11-12) ◽  
pp. 397-401 ◽  
Author(s):  
A. Campbell ◽  
H. Smith
Keyword(s):  
Water Samples ◽  
Immunomagnetic Separation ◽  
Clean Water ◽  
Recovery Efficiency ◽  
Blue Book ◽  
Negative Results ◽  
Cryptosporidium Oocysts ◽  
Comparison Of Techniques ◽  
User Friendly ◽  
Multiple Samples

A prototype immunomagnetic separation (IMS) technique was tested in five laboratories, which undertake routine analysis of water samples for Cryptosporidium oocysts, by comparing the recovery efficiency of the IMS technique with techniques in current use (the “Blue Book” Standing Committee of Analysts, SCA, method and flow cytometry, FCM). In very low turbidity samples (clean waters) of both 1ml and 10ml volumes the IMS demonstrated significantly better results than both SCA and FCM methods. Not only were higher oocyst recovery efficiencies reported but variation in recovery efficiency was reduced and fewer negative results were reported from oocyst-seeded samples than with the other two techniques. In trials with clean water or low turbidity water, FCM was the technique which most consistently reported negative results in oocyst-seeded samples and for clean water this difference was found to be statistically significant. When the water sample was turbid the recovery efficiency of the IMS technique diminished. The results suggest that the IMS technique is affected to different extents by different material constituents in water concentrates and that FCM is apparently less affected by interfering particulate matter. Despite the potential difficulties with the IMS method with turbid water samples, the results from these trials indicate that this technique would be a very useful addition to the armoury of methods for the concentration of oocysts from water samples and was considered by the trial participants to be simple, user-friendly and applicable to the processing of multiple samples simultaneously.

Occurrence and Removal of Cryptosporidium spp. Oocysts and Giardia spp. Cysts in Kenyan Waste Stabilisation Ponds

1993 ◽  
Vol 27 (3-4) ◽  
pp. 97-104 ◽  
Author(s):  
A. M. Grimason ◽  
H. V. Smith ◽  
W. N. Thitai ◽  
P. G. Smith ◽  
M. H. Jackson ◽  
...  
Keyword(s):  
Waste Water ◽  
Water Samples ◽  
Laboratory Experiments ◽  
Municipal Waste ◽  
Retention Period ◽  
Waste Stabilisation Ponds ◽  
Cryptosporidium Spp ◽  
Physico Chemical ◽  
Microbiological Parameters ◽  
Final Effluent

This study was designed to determine tlie occurrence and removal of Cryptosporidium spp. oocysts and Giardia spp. cysts, in municipal waste-water by waste stabilisation ponds in tlie Republic of Kenya. Eleven waste stabilisation pond systems located in towns across Kenya were included. A total of 66 waste-water samples were examined for the presence of oocysts and cysts, comprising 11 raw waste-water and 55 pond effluent samples over a two month period. Cryptosporidium spp. oocysts were detected in 6 and Giardia spp. cysts in 9 of the designated pond systems analysed demonstrating their ubiquitous nature throughout Kenya. Oocyst levels detected in raw waste-water samples ranged from 12.5 - 72.97 oocysts/l and various pond effluents between 2.25 - 50 oocysts/l. Cyst levels detected in raw waste-water samples ranged from 212.5 to 6212.5 cysts/l and in various pond effluents from 3.125 to 230.7 cysts/l. No Cryptosporidium spp. oocysts were detected in the final effluent from any pond systems studied (11/11). Whereas no Giardia spp. cysts were detected in the final effluent from 10 of 11 waste stabilisation pond systems studied, one pond system was found to be consistently discharging cysts in the final effluent at concentrations ranging from 40 to 50 cysts/l. The minimum retention period for the removal of Cryptosporidium spp. oocysts and Giardia spp. cysts was 37.3 days. Laboratory experiments were performed to assess physico-chemical and microbiological parameters to express relationships between pond performance and protozoa removal.

Enumeration of Cryptosporidium oocysts from surface water concentrates by laser-scanning cytometry

2000 ◽  
Vol 41 (7) ◽  
pp. 197-202 ◽  
Author(s):  
F. Zanelli ◽  
B. Compagnon ◽  
J. C. Joret ◽  
M. R. de Roubin
Keyword(s):  
Surface Water ◽  
Water Samples ◽  
Laser Scanning ◽  
Immunomagnetic Separation ◽  
Entire Surface ◽  
Cryptosporidium Oocysts ◽  
Different Types ◽  
Laser Scanning Cytometry ◽  
Direct Microscopic Observation

The utilization of the ChemScan® RDI was tested for different types of water concentrates. Concentrates were prepared by cartridge filtration or flocculation, and analysed either without purification, or after Immunomagnetic separation (IMS) or flotation on percoll-sucrose gradients. Theenumeration of the oocysts was subsequently performed using the ChemScan® RDI Cryptosporidium application. Enumeration by direct microscopic observation of the entire surface of the membrane was carried out as a control, and recoveries were calculated as a ratio between the ChemScan® RDI result and the result obtained with direct microscopic enumeration. The Chemscan enumeration technique proved reliable, with recoveries yielding close to 100% in most cases (average 125%, range from 86 to 467%) for all the concentration/purification techniques tested. The quality of the antibodies was shown to be critical, with antibodies from some suppliers yielding recoveries a low as 10% in some cases. This difficulty could, however, be overcome by the utilization of the antibody provided by Chemunex. These data conclusively prove that laser scanning cytometry, which greatly facilitates the microscopic enumeration of Cryptosporidium oocysts from water samples and decreases the time of observation by four to six times, can be successfully applied to water concentrates prepared from a variety of concentration/purification techniques.

Determination of the Recovery Efficiency of Cryptosporidium Oocysts and Giardia Cysts from Seeded Bivalve Mollusks

2013 ◽  
Vol 76 (1) ◽  
pp. 93-98 ◽  
Author(s):  
FRANCISKA M. SCHETS ◽  
HAROLD H. J. L. van den BERG ◽  
ANA MARIA de RODA HUSMAN
Keyword(s):  
Quantitative Assessment ◽  
Intestinal Parasites ◽  
Cryptosporidium Oocyst ◽  
Immunomagnetic Separation ◽  
Giardia Cyst ◽  
Bivalve Mollusks ◽  
Recovery Efficiency ◽  
Risk Of Infection ◽  
Cryptosporidium Oocysts

The intestinal parasites Cryptosporidium and Giardia are transmitted by water and food and cause human gastroenteritis. Filter-feeding bivalve mollusks, such as oysters and mussels, filter large volumes of water and thus concentrate such pathogens, which makes these bivalves potential vectors of disease. To assess the risk of infection from consumption of contaminated bivalves, parasite numbers and parasite recovery data are required. A modified immunomagnetic separation (IMS) procedure was used to determine Cryptosporidium oocyst and Giardia cyst numbers in individually homogenized oysters (Crassostrea gigas) and mussels (Mytilus edulis). About 12% of the commercial bivalves were positive, with low (oo)cyst numbers per specimen. The recovery efficiency of the IMS procedure was systematically evaluated. Experiments included seeding of homogenized bivalves and whole animals with 100 to 1,000 (oo)cysts. Both seeding procedures yielded highly variable recovery rates. Median Cryptosporidium recoveries were 7.9 to 21% in oysters and 62% in mussels. Median Giardia recoveries were 10 to 25% in oysters and 110% in mussels. Giardia recovery was significantly higher than Cryptosporidium recovery. (Oo)cysts were less efficiently recovered from seeded whole animals than from seeded homogenates, with median Cryptosporidium recoveries of 5.3% in oysters and 45% in mussels and median Giardia recoveries of 4.0% in oysters and 82% in mussels. Both bivalve homogenate seeding and whole animal seeding yielded higher (oo)cyst recovery in mussels than in oysters, likely because of the presence of less shellfish tissue in IMS when analyzing the smaller mussels compared with the larger oysters, resulting in more efficient (oo)cyst extraction. The data generated in this study may be used in the quantitative assessment of the risk of infection with Cryptosporidium or Giardia associated with the consumption of raw bivalve mollusks. This information may be used for making risk management decisions.

scholarly journals Investigation of Cryptosporidium spp. and Giardia spp. in Lake Eğirdir (Isparta)

2021 ◽  
Author(s):  
Tuğba Sağlam ◽  
Serdar Düşen ◽  
Meral Apaydın Yağcı ◽  
Abdülkadir Yağcı
Keyword(s):  
Public Health ◽  
Drinking Water ◽  
Water Samples ◽  
Average Density ◽  
Agricultural Irrigation ◽  
Control Programs ◽  
Cryptosporidium Spp ◽  
Direct Microscopic Examination ◽  
Different Seasons ◽  
Autumn And Winter

Objective: The aim of this study was to assess both the presence and seasonal variability of Cryptosporidium spp. and Giardia spp. in Eğirdir Lake within the borders of Isparta province, which is used for drinking, agricultural irrigation and recreational purposes. Method: The research was carried out between July 2016 and January 2017 and water samples were taken from five different stations in three different seasons in Lake Eğirdir. After direct microscopic examination of the samples (Native-Lugol method), they were stained with Modified Acid Fast (MAF), and examined under the light microscope for parasites. Results: Cryptosporidium spp and Giardia spp were detected in 15 water samples in summer months, with an average density of 99.2% and 93.3% respectively, in Lake Eğirdir. In addition, both parasites were also detected intensively in autumn and winter Conclusion: The use of Lake Eğirdir for daily needs of people, agriculture andrecreational purposes cause increase in protozoal density. Thus, it is necessary to conduct parasitological studies on Lake Eğirdir, especially during the periods of swimming tourism, to determine the protozoal epidemiology in humans and animals. In addition, it is important to carry out adequate disinfection processes and plan the necessary control programs in terms of public health in the regions where Lake Eğirdir is used as drinking water.

scholarly journals Detection and Discrimination of Cryptosporidium parvum and C. hominis in Water Samples by Immunomagnetic Separation-PCR

2005 ◽  
Vol 71 (2) ◽  
pp. 898-903 ◽  
Author(s):  
Yoshitsugu Ochiai ◽  
Chieko Takada ◽  
Mitsugu Hosaka
Keyword(s):  
Cryptosporidium Parvum ◽  
Water Samples ◽  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Detection Method ◽  
Fluorescent Antibody ◽  
Immunomagnetic Separation ◽  
Antibody Assay ◽  
Pcr Products ◽  
Genetic Detection

ABSTRACT Cryptosporidium parvum and C. hominis have been the cause of large and serious outbreaks of waterborne cryptosporidiosis. A specific and sensitive recovery-detection method is required for control of this pathogen in drinking water. In the present study, nested PCR-restriction fragment length polymorphism (RFLP), which targets the divergent Cpgp40/15 gene, was developed. This nested PCR detected only the gene derived from C. parvum and C. hominis strains, and RFLP was able to discriminate between the PCR products from C. parvum and C. hominis. To evaluate the sensitivity of nested PCR, C. parvum oocysts inoculated in water samples of two different turbidities were recovered by immunomagnetic separation (IMS) and detected by nested PCR and fluorescent antibody assay (FA). Genetic detection by nested PCR and oocyst number confirmed by FA were compared, and the results suggested that detection by nested PCR depends on the confirmed oocyst number and that nested PCR in combination with IMS has the ability to detect a single oocyst in a water sample. We applied an agitation procedure with river water solids to which oocysts were added to evaluate the recovery and detection by the procedure in environmental samples and found some decrease in the rate of detection by IMS.

scholarly journals Quantitative Detection of Legionella pneumophila in Water Samples by Immunomagnetic Purification and Real-Time PCR Amplification of the dotA Gene

2005 ◽  
Vol 71 (7) ◽  
pp. 3433-3441 ◽  
Author(s):  
M. A. Yáñez ◽  
C. Carrasco-Serrano ◽  
V. M. Barberá ◽  
V. Catalán
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Legionella Pneumophila ◽  
Water Samples ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Immunomagnetic Separation ◽  
Pcr Analysis ◽  
Quantitative Detection ◽  
Specific Sequence

ABSTRACT A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.

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