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

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