(Eh) y metanÃ³lico (Em) de Pera distichophylla sobre un aislado de ...

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A. da S. BARBOSA et al. animals as the parameter of choice, totaling 16 soil samples collected for each collection point. The water from surface sources (raw water) was collected by means of the discharge of 2000 liters in commercial Aqualimp® filters, having blanket cartridges and coiled wire with 1 µm porosity of Cuno® DPPPY-1 Micro Wind II model. After filtration, the cartridges were packaged in properly marked plastic bags. Around two liters of reservoir-bottom sediments were collected from two polyethylene reservoirs (treated water) with the aid of a 1’ plastic hose through siphoning. This sediment was stored in 2 liter plastic bottles, previously cleaned and identified. In the soil, the collections were standardized in pool system, which represents the points surrounding each place previously determined as reference. Each collection point is 150 cm distant from the reference, and the collection points are 250 cm equidistant from each other. All the soil collected from the chosen point was stored in the same plastic bag, forming one sample. The material collected was obtained by surface scraping of the soil with a stainless steel paddle inside a square of 25 cm 2 , and a template was used. After the collection, soil samples were packaged in properly marked plastic bags. The water and soil samples were stored in refrigerated isothermal container and sent to the Parasitology Laboratory of the Biomedical Institute, at Federal Fluminense University (UFF) and then sent to the Immunodiagnostic Laboratory of the Department of Biological Sciences (DCB), at the National School of Public Health (ENSP)/FIOCRUZ. Preprocessing:The filter cartridges were eluted by washing with one liter of neutral detergent solution at 0.001% (q.s.p.), gauze filtered and placed for sedimentation. The samples stored in 2-liter plastic bottles were filtered and placed for sedimentation. In the soil, 100 g of each sample were used by deposit in conical bottom cups with 1-liter detergent solution at 0.001% (q.s.p.), and the material was homogenized and filtered with a gauze and placed for sedimentation. Surveys of protozoa cysts and helminth eggs using microscopic techniques: The sediment was aliquoted and all samples were subject to survey of evolutionary forms of protozoa and helminths. One mililiter of the sediment was used for each microscopic concentration technique. Three glass 92 slides per sample were read in Ritchie’s technique modified by Young et al, whereas one glass slide per sample was read in Sheather’s technique modified by Huber et al. All slides were covered with a 24 x 32 mm coverslip, and viewing and photomicrography were made on a Olympus® Bx 41 binocular optical microscope with an increase of approximately 100 times and confirmation in 400 times, coupled to a Samsung® SDC415 digital camera, complete with Honestech® PVR capture software. For morphometry, we used an Olympus® CH30 monocular optical microscope with 400 times magnification, and an ocular micrometer. Survey of surface antigens via Immunodiagnosis: The Enzyme-linked Immunosorbent Assay, ELISA, was conducted in all samples for survey of G. lamblia, Cryptosporidium sp., and E. histolytica. The kits were purchased commercially: Techlab ® Wampole Cryptosporidium II test, which surveys the antigens of the oocyst wall of Crypstosporidium sp.; Entamoeba Ridascreen ® , which surveys adhesins specific of E. histolytica (N-acetyl-D-galactosamine lectin inhibitor), present on the parasite membrane; and Giardia Ridascreen ® , which surveys antigens specific of the cyst wall of G. lamblia. Before the performance of ELISA, a predilution of 60 μL of the sample and 60 μL of the diluent provided by manufacturer was performed, and 100 μL of this solution was transferred to the duplicate test plate. Then, we followed the protocol recommended by the manufacturer. The reading was made in ELISA reader (Testline® ELx 800) with wavelength according to the manufacturer’s technical standards. Statistical treatment of results: The McNemar’s statistical test with a significance level of 5% was used with comparison degree of agreement between the laboratory techniques, using SPSS® (Statistical Package for Social Sciences, Chicago), of version 17.0. RESULTS In the 48 water samples examined, was observed agreement applying Sheather’s modified and Ritchie’s modified techniques to detect the amoeboid cyst with more than four nuclei, four- Rev. Ibero-Latinoam. Parasitol. (2012); 71 (1): 90-96
nucleus amoeboid cyst and cyst of Giardia sp. in the same samples. It was not possible to compare the degree of agreement between these techniques in the survey of the oocyst of Cystoisospora sp., nematode larvae and eggs, coccidia oocysts and eggs of Trichuris sp., as these evolutionary forms were detected only in one of the techniques and, for comparison using McNemar’s test, it must be positive at least once in the two of them (Table 1). In water, ELISA detected more positive samples for the protozoa G. lamblia, E. histolytica e Cryptosporidium sp. than the microscopic Ritchie’s and Sheather’s modified techniques. Despite the greater positivity in ELISA, it was still possible to find the degree of agreement with the microscopic techniques. This agreement happened in the detection of four-nucleus amoeboid cysts in two samples with sizes compatible with E. histolytica/E. dispar complex, confirmed for E. histolytica in ELISA, cysts of Giardia sp. in four samples with dimensions compatible with G. lamblia, confirmed with ELISA. Comparing the degree of agreement in the survey of Cryptosporidium sp. was not possible, as the result was possible only in ELISA (Table 2). In the 64 soil samples collected, there was agreement between the microscopic techniques Table 1. Comparison between the results obtained with Sheather’s modified and Ritchie’s modified techniques for detection protozoa and helminths in 48 water samples Protozoa and helminthes Ritchie’s mod. technique Sheather’s mod. technique MN No+ No - No+ No - SxR Amoeboid cyst with more than 4 nuclei 2 46 4 44 0.5 Four-nucleus amoeboid cyst 2 46 2 46 1 Cyst of Giardia sp. 4 44 4 44 1 Cyst of Cystoisospora sp. 0 48 1 47 NF Nematode larvae 4 44 0 48 NF Nematode egg 0 48 3 45 NF Coccidia oocyst 0 48 1 47 NF Egg of Trichuris sp. 0 48 1 47 NF No+: Number of positive samples; No-: Number of negative samples; R: Ritchie’s technique modified by Young et al.; S: Sheather’s technique modified by Huber et al.; NF: Comparison was not possible; MN: McNemar Test. Table 2. Comparison between the results obtained with Ritchie’s modified technique, Sheather’s modified technique and ELISA for detection of the protozoa Giardia lamblia, Entamoeba histolytica and Cryptosporidium sp. in 48 water samples Protozoa ELISA Ritchie’s mod. technique Sheather’s mod. technique No.+ No.- No.+ No.- No.+ No.- ExR ExS Cyst of Entamoeba histolytica 6 42 2 46 2 46 0.08 0.08 Oocyst of Cryptosporidium sp. 10 38 0 48 0 48 NF NF Cyst of Giardia lamblia 9 39 4 44 4 44 0.06 0.06 No.+: Number of positive samples; No.-: Number of negative samples; E: ELISA; R: Ritchie’s technique modified by Young et al.; S: Sheather’s technique modified by Huber et al.; NF: Comparison was not possible; MN: McNemar Test. Rev. Ibero-Latinoam. Parasitol. (2012); 71 (1): 90-96 COMPARISON OF TECHNIQUES IN ENVIRONMENTAL SAMPLES MN 93
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CONTINUACIÓN DE PARASITOLOGÍA LAT
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Producción: María Cristina Illane
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Contenido 4 Rev. Ibero-Latinoam. Pa
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M. T. GALáN-PUCHADES and A. OSUNA
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Artículo Original 14 Rev. Ibero-La
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A. MUÑOZ et al. cionalmente, exist
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C. ZÚÑIGA et al. RESUMEN Ratones
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