Detection Of Giardia Cysts Research Articles

A comparison of phase contrast microscopy and an immunofluorescence test for the detection of Giardia spp. in faecal specimens from cattle and wild rodents

As part of a study on the presence of Giardia cysts in water, a commercial direct immunofluorescence (DIF) test for the cysts was evaluated and compared to phase contrast microscopy. Forty faecal samples were collected from cattle; 31 contained Giardia. All 31 samples were positive by DIF whereas only 17 were detected by microscopy. The detected Giardia cysts were identified as belonging to the G. duodenalis group. 216 faecal samples were collected from wild rodents ( Apodemus flavicollis, A. sylvaticus and Clethrionomys glareolus); 103 contained Giardia. Cysts were detected in 97 samples by DIF and in 57 by microscopy; 51 samples were positive by both methods. Nineteen rodents harboured cysts which morphologically resembled G. duodenalis. Specific identification was possible only by phase contrast microscopy, but the results suggested that DIF was superior for the detection of Giardia cysts in faeces of the animals tested.

Identification of algae which interfere with the detection of Giardia cysts and Cryptosporidium oocysts and a method for alleviating this interference.

Fifty-four algal species were tested for cross-reaction in the American Society for Testing and Materials Giardia/Cryptosporidium indirect immunofluorescence assay, and 24 showed some degree of fluorescence. Two species, Navicula minima and Synechococcus elongatus, exhibited a bright apple green fluorescence. The addition of goat serum to the assay mixture blocked the fluorescence of most nontarget organisms tested and also decreased the background fluorescence. Goat serum did not interfere with the fluorescence of Giardia cysts or Cryptosporidium oocysts or the identification of cyst and oocyst internal structures.

Comparison of two methods for detection of Giardia cysts and Cryptosporidium oocysts in water

The steps of two immunofluorescent-antibody-based detection methods were evaluated for their efficiencies in detecting Giardia cysts and Cryptosporidium oocysts. The two methods evaluated were the American Society for Testing and Materials proposed test method for Giardia cysts and Cryptosporidium oocysts in low-turbidity water and a procedure employing sampling by membrane filtration, Percoll-Percoll step gradient, and immunofluorescent staining. The membrane filter sampling method was characterized by higher recovery rates in all three types of waters tested: raw surface water, partially treated water from a flocculation basin, and filtered water. Cyst and oocyst recovery efficiencies decreased with increasing water turbidity regardless of the method used. Recoveries of seeded Giardia cysts exceeded those of Cryptosporidium oocysts in all types of water sampled. The sampling step in both methods resulted in the highest loss of seeded cysts and oocysts. Furthermore, much higher recovery efficiencies were obtained when the flotation step was avoided. The membrane filter method, using smaller tubes for flotation, was less time-consuming and cheaper. A serious disadvantage of this method was the lack of confirmation of presumptive cysts and oocysts, leaving the potential for false-positive Giardia and Cryptosporidium counts when cross-reacting algae are present in water samples.

Evaluation of the immunofluorescence procedure for detection of Giardia cysts and Cryptosporidium oocysts in water.

The accurate determination of the presence of Giardia cysts and Cryptosporidium oocysts in surface waters requires a reliable method for the detection and enumeration of these pathogenic organisms. Published methods have usually reported recovery efficiencies of less than 50% for both cysts and oocysts. Typically, the losses are greater for Cryptosporidium oocysts than they are for Giardia cysts. The purpose of this study was to examine procedures used for sample collection, elution, concentration, and clarification to determine when losses of cysts and oocysts occurred during processing. The results showed that major losses of cysts and oocysts occurred during centrifugation and clarification. Depending on the centrifugation force, oocyst losses of as high as 30% occurred for each centrifugation step. A 1.15-specific-gravity Percoll-sucrose gradient was needed to optimize recovery of oocysts from natural water samples. Minor improvements in the procedure could be accomplished by selecting a filter other than the recommended 1-micron-pore-size (nominal-porosity) polypropylene filter.

Evaluation of a new monoclonal antibody combination reagent for direct fluorescence detection of Giardia cysts and Cryptosporidium oocysts in human fecal specimens.

Giardia lamblia and Cryptosporidium parvum can cause severe symptoms in humans, particularly in the immunologically compromised. Monoclonal antibody reagents offer increased sensitivity and an excellent alternative to conventional staining methods. These reagents are helpful when screening large numbers of patients or those with minimal symptoms. Problems of false-positive and false-negative results with routine staining methods for stool parasites can be eliminated with monoclonal antibody reagents. Known positive formalinized specimens [Giardia sp. (n = 60), Cryptosporidium sp. (n = 55), and mixed Giardia-Cryptosporidium spp. (n = 10)] and negative formalinized specimens (n = 105), of which 46 contained other yeast or human cells or protozoa), were tested by the MERIFLUOR Cryptosporidium-Giardia direct immunofluorescence detection procedure. The MERIFLUOR reagent exhibited +/- to 4+ (majority, 2+ to 3+) on all Giardia cysts and 2+ to 4+ (majority, 3+ to 4+) on all Cryptosporidium oocysts. The cysts were generally oval (11 to 15 microns), while the oocysts were round (4 to 6 microns); both showed apple-green fluorescence against a background free of nonspecific fluorescence. All specimens positive for Giardia sp. and/or Cryptosporidium sp. showed fluorescence, and all specimens negative for the two organisms showed no fluorescence. There were eight specimens previously negative by the ova and parasite examination which were positive by the direct fluorescence method; four contained Giardia sp., and four contained Cryptosporidium sp. These positive results were confirmed after the examination of additional trichrome and modified acid-fast smears. The MERIFLUOR reagent was very easy to use, and even with a lower fluorescence intensity for Giardia sp. cysts, no false-negative or false-positive results among the specimens tested for either organism were found.

Studies on the giardiasis as the zoonosis. III. Prevalence of Giardia among the dogs and the owners in Japan

To obtain the basic data on the route of Giardia infection as zoonosis, of many regions in Japan, the feces from 2218 dogs were examined for detection of Giardia cysts. Giardia cysts were detected in 239 of the 2218 dogs (10.9%), which was the same as previous reports from America. None were found from the owners of 51 dogs in which Giardia cysts were detected. The detection rates of each facilities were, 68 of 366 (18.6%) from the breeder's kennels, 169 of 1811 (9.3%) in individual houses, 2 of 42 (4.9%) from research institutes. The detection rate of the breeder's kennels was higher than the other two facilities (p less than 0.05, p less than 0.001). The detection rates of Kanagawa and Shizuoka prefectures among 17 regions in Japan were higher than the others (p less than 0.001, p less than 0.05). Especially in Shizuoka, the rate of the individual houses was higher than from breeder's kennels. In kanagawa the rates of the individual houses and the breeders kennels were higher than the mean in Japan (p less than 0.001). Therefore one must instruct the breeders when teaching health education to included zoonosis, and that the detection rate of the age groups of less than 3 years old was high-221 of 1276 (17.3%). Since the detection rates of Giardia cysts in the dogs were low, the possibility that human infection acquired from dogs was low. However, some of patients with giardiasis we encountered had never been abroad, and it is not yet clear whether Giardia is strictly host specific or not, so attention should be paid to the possibility of cross-infection between man and animals.

Detection of Giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts

A method was developed for the detection of Giardia cysts by using the polymerase chain reaction (PCR) and the giardin gene as the target. DNA amplification by PCR, using giardin DNA as the target, resulted in detection of both live and dead cysts. When giardin mRNA was used as the target, the ability to amplify cDNA by PCR depended on the mode of killing. Cysts killed by freezing were not detected by PCR when giardin mRNA was the target. Cysts killed by heating or exposure to monochloramine, however, gave positive detection signals for both DNA and giardin mRNA targets. The amount of giardin mRNA and total RNA was significantly increased in live cysts following the induction of excystation. Cysts killed by freezing, heating, or exposure to monochloramine did not show a change in RNA content. The detection of the giardin gene by PCR permits a sensitive and specific diagnosis for Giardia spp. Discrimination between live and dead cysts can be made by measuring the amounts of RNA or PCR-amplified product from the giardin mRNA target before and after the induction of excystation.

Detection of Giardia cysts with a cDNA probe and applications to water samples

Giardiasis is the most common human parasite infection in the United States, causing a lengthy course of diarrhea. Transmission of Giardia species is by the fecal-oral route, and numerous waterborne outbreaks have been documented. The Environmental Protection Agency has regulated Giardia species in drinking water through the Surface Water Treatment Rule. Current methods for detection of Giardia species in water rely primarily on microscopic observation of water concentrates with immunofluorescence techniques. We evaluated the efficacy of using a gene-specific probe for the detection of Giardia species in water. A cDNA probe, 265 bp long, from the small subunit of rRNA of Giardia lamblia was used for detection of cysts. The replicative form of the M13 vector with an insert was isolated from lysed host Escherichia coli XL1-Blue and used for production of the cDNA probe by nick translation with 32P-labeled nucleotides. Six different protocols were tested for extracting nucleic acids from the cysts. With the most efficient procedure, disrupting Giardia cysts with glass beads in the presence of proteinase K, as few as 1 to 5 cysts per ml can be detected in water sample concentrates with dot blot hybridization assays.

A Comparison of Giardia microti and Spironucleus muris Cysts in the Vole: An Immunocytochemical, Light, and Electron Microscopic Study

We have shown that cysts of the genus Spironucleus share many common morphological features with Giardia cysts including: 2-4 nuclei, flagellar axonemes, a distinct cyst wall, and they even display the same immunostaining as Giardia cysts when labeled with antibodies specific for Giardia cyst wall. A direct comparison of Spironucleus muris and Giardia microti cysts have revealed that cysts of S. muris are significantly smaller than cysts of G. miroti. At the ultrastructural level, the cyst walls are similar in fibrillar appearance, but the width of the S. muris cyst wall is significantly less than that of G. microti. The cysts of S. muris also differ from G. microti in that they contain a striated rootlet fiber, flagellar sheath, and numerous glycogen rosettes. Characteristic features of Giardia include the adhesive disc and median body. Although the cysts of Spironucleus and Giardia are similar in appearance, these unique morphological features can be used to distinguish between the 2 protozoa and should be employed in the detection of Giardia cysts in water samples.

ISOLATION AND DETECTION OF GIARDIA CYSTS FROM WATER USING DIRECT IMMUNOFLUORESCENCE1

ABSTRACT: A water‐sampling apparatus used for the isolation and detection of Giardia cysts in water has been designed and tested. The sampling apparatus uses one of a variety of pumps or waterline pressure to move water through a filter. Two of the optional pumps are lightweight enough to make the apparatus portable and thus suitable for sampling in remote areas. This technique of sample processing produces good cyst recovery in much less time than is required with previously established methods. Giardia cysts are identified using direct immunofluorescence.

Evaluation of a tangential-flow multiple-filter technique for detection of Giardia lamblia cysts in water.

A system of tangential-flow filtration was evaluated for use in the detection of Giardia cysts in drinking water. This method was more sensitive in recovering cysts than a frequently used wound-orlon system of through-filtration.

Detection of Giardia cysts at low concentrations in water using nuclepore membranes

A filtration procedure was devised to recover Giardia cysts from water using 5 μm Nuclepore membranes (110 mm in dia). Cysts taken from a beaver ( Castor canadensis) were added to 100 l. of untreated stream water and recovered by filtration. Cysts were washed from the membrane, concentrated by centrifugation and microscopically examined. Recovery efficiencies averaged 53% at cyst concentrations between 0.5 and 45 cysts l −1. Maximum cyst recovery was observed at filtration pressures of 40–60 kPa. The advantages offered by this method over cartridge filtration methods include higher recovery efficiencies at low cyst concentrations and simpler, more rapid laboratory procedures.