New method using sedimentation and immunomagnetic separation for isolation and enumeration of Cryptosporidium parvum oocysts and Giardia lamblia cysts.
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A new method for the isolation of Cryptosporidium parvum oocysts and Giardia lamblia cysts from biosolid samples has been developed that utilizes sedimentation and immunomagnetic separation. The method was used to recover stained cysts and oocysts (spike organisms) from primary settled sewage sludge, anaerobically digested sewage sludge, and bovine manure. Recovery efficiencies associated with this method were approximately 40 to 60% and were significantly greater than those associated with similar methods based on sucrose flotation (P < 0.001). The recovery efficiency of the sedimentation-based method showed no significant reduction as a result of sample storage for up to 21 days (P > 0.05). Recovery efficiencies were determined by spiking samples with prestained cysts and oocysts, allowing them to be differentiated from those naturally present in the biosolid samples. The prestained cysts and oocysts had been fixed in 5% formalin, and the recovery efficiencies associated with this method may be different from recovery efficiencies for fresh cysts or oocysts. (+info)
Methodology of protistan discovery: from rRNA detection to quality scanning electron microscope images.
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Each year, thousands of new protistan 18S rRNA sequences are detected in environmental samples. Many of these sequences are molecular signatures of new protistan species, classes, and/or kingdoms that have never been seen before. The main goal of this study was to enable visualization of these novel organisms and to conduct quality ultrastructural examination. We achieved this goal by modifying standard procedures for cell fixation, fluorescence in situ hybridization, and scanning electron microscopy (SEM) and by making these methodologies work in concert. As a result, the same individual cell can now be detected by 18S rRNA-targeted fluorochrome-labeled probes and then viewed by SEM to reveal its diagnostic morphological characteristics. The method was successfully tested on a wide range of protists (alveolates, stramenopiles, kinetoplastids, and cryptomonads). The new methodology thus opens a way for fine microscopy studies of many organisms previously known exclusively by their 18S rRNA sequences. (+info)
Evaluation of the Abbott Cell-Dyn 4000 hematology analyzer for detection and therapeutic monitoring of Plasmodium vivax in the Republic of Korea.
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The Cell-Dyn 4000 automated hematology analyzer (CD4000) has the ability to detect malaria patients, but it remained unclear whether it could detect persistent malaria post-treatment. To investigate this, we used the CD4000 to evaluate 68 Korean patients with Plasmodium vivax malaria, and control groups of 50 patients with fever and 50 asymptomatic patients. The results from the instrument-generated scatter plot (derived by laser light depolarization) were compared with microscopy results. During the initial diagnosis, the sensitivity of the CD4000 in detecting malaria was 91.2%. On day 3 of follow-up, the CD4000 results matched those from microscopy by 96.7%. Malaria was not detected by either method beyond 14 days post-presentation. Interestingly, the atypical depolarizing events, which typify the presence of malaria in the analyzer, were highly correlated with the levels of parasitaemia in serially diluted samples of the leucocyte-depleted blood, and the CD4000 detected parasites down to the level of 288 +/- 17.7/microl. Our findings suggest that the phenomenon of atypical light depolarization could be influenced by parasitaemia levels, and be used as a screening method for P. vivax malaria patients, as well as for the therapeutic monitoring. (+info)
External quality assessment schemes raise standards: evidence from the UKNEQAS parasitology subschemes.
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BACKGROUND: The burden of parasitic disease imported into the temperate zone is increasing, and in the tropics remains very high. Thus, high quality diagnostic parasitology services are needed, but to implement clinical governance a measure of quality of service is required. AIM: To examine performance in the United Kingdom National External Quality Assessment Scheme for Parasitology for evidence of improved standards in parasite diagnosis in clinical specimens. METHODS: Analysis of performance was made for the period 1986 to 2001, to look for trends in performance scores. RESULTS: An overall rise in performance in faecal and blood parasitology schemes was found from 1986 to 2001. This was seen particularly in the identification of ova, cysts, and larvae in the faecal scheme, the detection of Plasmodium ovale and Plasmodium vivax in the blood scheme, and also in the correct identification of non-malarial blood parasites. Despite this improvement, there are still problems. In the faecal scheme, participants still experience difficulty in recognising small protozoan cysts, differentiating vegetable matter from cysts, and detecting ova and cysts when more than one species is present. In the blood scheme, participants have problems in identifying mixed malarial infections, distinguishing between P ovale and P vivax, and estimating the percentage parasitaemia. The reasons underlying these problems have been identified via the educational part of the scheme, and have been dealt with by distributing teaching sheets and undertaking practical sessions. CONCLUSIONS: UK NEQAS for Parasitology has helped to raise the standard of diagnostic parasitology in the UK. (+info)
In vitro induction of Neospora caninum bradyzoites in vero cells reveals differential antigen expression, localization, and host-cell recognition of tachyzoites and bradyzoites.
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We report on an optimized method for the in vitro culture of tissue cyst-forming Neospora caninum bradyzoites in Vero cells and the separation of viable parasites from host cells. Treatment of tachyzoite-infected Vero cell cultures with 17 microM sodium nitroprusside for 8 days severely scaled down parasite proliferation, led to reduced expression of tachyzoite surface antigens, and induced the expression of the bradyzoite marker NcBAG1 and the cyst wall antigen recognized by the monoclonal antibody MAbCC2. Transmission electron microscopy demonstrated that intracellular parasites were located within parasitophorous vacuoles that were surrounded by a cyst wall-like structure, and the dense granule antigens NcGRA1, NcGRA2, and NcGRA7 were incorporated into the cyst wall. Adhesion-invasion assays employing purified tachyzoites and bradyzoites showed that tachyzoites adhered to, and invaded, Vero cells with higher efficiency than bradyzoites. However, removal of terminal sialic acid residues from either the host cell or the parasite surface increased the invasion of Vero cells by bradyzoites, but not tachyzoites. (+info)
Comparison between immunomagnetic separation, coupled with immunofluorescence, and the techniques of Faust et al. and of Lutz for the diagnosis of Giardia lamblia cysts in human feces.
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In the present study, the performance of Immunomagnetic Separation technique, coupled with Immunofluorescence (IMS-IFA), was compared with the FAUST et al. and Lutz parasitological techniques for the detection of Giardia lamblia cysts in human feces. One hundred and twenty-seven samples were evaluated by the three techniques at the same time showing a rate of cyst detection of 27.5% by IMS-IFA and 15.7% by both Faust et al. and Lutz techniques. Data analysis showed a higher sensitivity of IMS-IFA for the detection of G. lamblia cysts in comparison with the techniques of FAUST et al. and Lutz. The use of this methodology as a routine procedure enables the processing of many samples simultaneously, in order to increase recovery rate of G. lamblia cysts and reduce the time of sample storage. (+info)
Evaluation of five membrane filtration methods for recovery of Cryptosporidium and Giardia isolates from water samples.
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We evaluated the efficiency of five membrane filters for recovery of Cryptosporidium parvum oocysts and Giardia lamblia cysts. These filters included the Pall Life Sciences Envirochek (EC) standard filtration and Envirochek high-volume (EC-HV) membrane filters, the Millipore flatbed membrane filter, the Sartorius flatbed membrane filter (SMF), and the Filta-Max (FM) depth filter. Distilled and surface water samples were spiked with 10 oocysts and 10 cysts/liter. We also evaluated the recovery efficiency of the EC and EC-HV filters after a 5-s backwash postfiltration. The backwashing was not applied to the other filtration methods because of the design of the filters. Oocysts and cysts were visualized by using a fluorescent monoclonal antibody staining technique. For distilled water, the highest percent recovery for both the oocysts and cysts was obtained with the FM depth filter. However, when a 5-s backwash was applied, the EC-HV membrane filter (EC-HV-R) was superior to other filters for recovery of both oocysts (n = 53 +/- 15.4 per 10 liters) and cysts (n = 59 +/- 11.5 per 10 liters). This was followed by results of the FM depth filter (oocysts, 28.2 +/- 8, P = 0.015; cysts, 49.8 +/- 12.2, P = 0.4260), and SMF (oocysts, 16.2 +/- 2.8, P = 0.0079; cysts, 35.2 +/- 3, P = 0.0079). Similar results were obtained with surface water samples. Giardia cysts were recovered at higher rates than were Cryptosporidium oocysts with all five filters, regardless of backwashing. Although the time differences for completion of filtration process were not significantly different among the procedures, the EC-HV filtration with 5-s backwash was less labor demanding. (+info)
PCR-based identification of zoonotic isolates of Blastocystis from mammals and birds.
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The genotype of Blastocystis isolated from humans and animals is highly polymorphic. Therefore, it is important to compare the genotypes of Blastocystis isolates from humans and animals to determine the zoonotic potential of animal isolates. PCR-based genotype classification using known sequence-tagged site (STS) primers allows identification of zoonotic isolates of animal origin. To this end, 51 isolates from monkeys, cattle, pigs, chickens, quails and pheasants were subjected to genotype analysis using seven kinds of STS primers. Out of the 51 isolates, 39 were identified as one of the known genotypes, four showed mixed genotypes, and eight were unknown genotypes as these were negative for all STS primers. When these results were combined with previous studies on 41 isolates from animals and compared with the diversity of genotypes of 102 human Blastocystis hominis isolates, 67.4 % (62/92) of isolates from mammals and birds were identical to human B. hominis genotypes. Since the unknown genotype of human origin had been placed into an additional clade in the small-subunit rRNA gene phylogeny, further molecular study on the eight isolates of unknown genotype from the present study will facilitate our understanding of their zoonotic potential. (+info)