RESEARCH NOTE/ NOTA CIENTÍFICA
DIAGNOSIS OF EXPERIMENTAL INFECTION BY ECHINOSTOMA PARAENSEI LIE
AND BASCH, 1967 (TREMATODA, ECHINOSTOMATIDAE)
DIAGNÓSTICO DE LA INFECCIÓN EXPERIMENTAL POR ECHINOSTOMA
PARAENSEI LIE Y BASCH, 1967 (TREMATODA, ECHINOSTOMATIDAE)
1 1 1 2
Vinicius M. Tunholi-Alves , Victor M. A. Tunholi , Sócrates Costa-Neto , Ludmila S Amaral ,
3 4 5
Arnaldo Maldonado Jr , Jairo Pinheiro & Maria de Lurdes de A. Rodrigues
1Programa de Pós-Graduação em Ciências Veterinárias, Deptº Parasitologia Animal, Instituto de Veterinária, UFRuralRJ;
2Programa de Pós-Graduação em Saúde Pública, Departamento de Saneamento e Saúde Ambiental, Escola Nacional de
3
Saúde Pública, Fiocruz, RJ; Laboratório de Biologia e Parasitologia de Mamíferos Silvestres Reservatórios, Instituto
4
Oswaldo Cruz, Fiocruz, RJ; Área de Biofísica, Departamento de Ciências Fisiológicas, Instituto de Biologia,
5
UFRuralR; Deptº de Parasitologia Animal, Inst.Veterinária, UFRuralRJ.- BR 465 Seropédica, RJ, 23890-000, Brazil.
mlazrod@uol.com.br
Neotropical Helminthology, 2015, 9(1), jan-jun: 171-176.
171
ISSN Versión impresa 2218-6425 ISSN Versión Electrónica 1995-1043
ABSTRACT
Keywords: Echinostoma paraensei - efficiency - Hoffman - Kato-Katz - sensitivity.
The diagnosis of infection by trematodes is essentially based on the microscopic observation of
eggs of parasites in feces, through qualitative and quantitative techniques. In this study, we
investigated the efficiency and sensitivity of the techniques of Kato-Katz and Hoffman used to
diagnose Echinostoma paraensei in infected and uninfected hamsters under experimental
conditions. The method of Hoffman showed the highest sensitivity and efficiency when
compared with the Kato-Katz method for the diagnosis of E. paraensei.
RESUMO
Palabras clave: Echinostoma paraensei - eficiencia - Hoffman - Kato -Katz - sensibilidad.
El diagnóstico de la infección por tremátodos se basa esencialmente en la observación
microscópica de los huevos de parásitos en las heces, a través de técnicas cualitativas y
cuantitativas. En este estudio, se investigó la eficacia y la sensibilidad de las técnicas de Kato-
Katz y Hoffman que se utilizan para diagnosticar Echinostoma paraensei en hámsters infectados
y no infectados en condiciones experimentales. El método de Hoffman mostró mayor
sensibilidad y eficacia en comparación con el método de Kato-Katz para el diagnóstico de E.
paraensei.
characterization of the dynamics of this
helminthiasis .
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Neotropical Helminthology. Vol. 9, Nº1, jan-jun 2015
INTRODUCTION
Trematodes diagnosis is essentially based on
the microscopic observation of eggs of
parasites in faeces, through qualitative and
quantitative techniques. The Kato-Katz
technique receives special attention, and is
mainly used in schistosomiasis control
programs, in addition to acting as an auxiliary
tool in the quantitative and qualitative
diagnosis of intestinal helminth infections
(Kato & Miura, 1954; Katz et al., 1972;
Feldmeier & Poggensee, 1993). The efficiency
and sensitivity of parasitological techniques
have been the focus of some studies (Táparo et
al., 2006). The Kato-Katz, widely used as a
standard for the diagnosis of Schistosoma
mansoni Sambon,1907, has shown over time,
the results of effectiveness (Santos et al., 2005)
when compared with the sedimentation
techniques (Martin & Beaver, 1968; Kongs et
al., 2001). Coproparasitologic techniques
results allow estimating the intensity of the
infection as main parameter for the evaluation
of the pathology, and prognosis response to
chemotherapy (Feldmeier & Poggensee,
1993). Adults of Echinostoma infect the small
intestine definitive host, in which we highlight
the waterfowl, mammals, including humans,
reptiles and fish (Lie & Basch, 1967; Fried &
Graczyk, 2000; Kanev et al., 2000; Maldonado
et al., 2001a, b;). Serological techniques and
molecular biology has been developed for the
diagnosis of different helminths (Barbosa
Campos et al., 1988; Graczyk & Fried, 1994),
but function as a complementary tool for
research studies, restricting its use in routine
laboratory because of the high cost of
implementation. For the first time, a study was
conducted comparing the techniques of Kato-
Katz and Hoffman in the diagnosis of
Echinostoma paraensei Lie & Basch, 1967 in
hamsters experimentally infected in order to
confirm the establishment of the fluke in
experimental and clinical studies, assisting
epidemiological studies focused on the
Tunholi-Alves et al.
MATERIAL AND METHODS
Samples
The cycle of E. paraensei is maintained in
experimental Laboratory of Parasitology of
Wild Mammals Reservoirs (LABPMR) of the
Oswaldo Cruz Institute (IOC), FIOCRUZ, Rio
de Janeiro, RJ, Brazil and faeces samples of 15
hamsters infected were collected with the aid
of plastic bags previously identified and kept
under refrigeration for twenty-four hours until
processing.
Parasitological Techniques
The experiment was conducted in LABPMR,
IOC, FIOCRUZ, Rio de Janeiro, RJ. The fecal
samples were homogenized, weighed and
processed in duplicate by the techniques of
Hoffman (Lutz, sedimentation) and Kato-Katz
(García et al., 1985).
Lutz (natural sedimentation)
Approximately one gram of faeces was
homogenized in water and the solution filtered
through a gaze to a bent sedimentation cup of
100 ml, and the volume completed with tap
water at room temperature. This suspension
allowed to stand for 30 min and then examined
under a light microscope using a 20x objective.
This is the routine technique used in
LABPMR.
Kato-Katz
One gram of feces was placed on absorbent
paper to remove excess moisture. Shortly
thereafter, a nylon fabric was compressed with
the aid of a spatula causing part of the stool
pass through its meshes. These were collected
with a spatula and pressed into the hole of a
perforated plate situated on a slide, until the
latter is full. The excess was removed with the
feces side of the spatula. Subsequently, the
perforated plate was removed, tilting initially
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Neotropical Helminthology. Vol. 9, Nº1, jan-jun 2015 Experimental infection by Echinostoma
one end so as to remain on the glass slide of a
cylinder fecal sample. About this cylinder was
placed a cellophane coverslip, previously
soaked in solution Diafix component of
commercial Kit Kato-Katz. The slide was then
inverted on a flat surface and pressed so as to
spread the material evenly between the slide
and coverslip cellophane preventing the
leakage of feces. Waiting for 30 min, to clarify
the fecal smear for t analysis in a light
microscope.
Statistical analyses
To evaluate the sensitivity, efficiency, number
of eggs recovered and to verify the normality
of the data the Kolmogorov Smirnoff analyses
were conducted. The results were expressed as
mean ± standard error and submitted to the
Tukey test for comparison of means (α = 5%).
The results indicated significant variations in
their sensitivity, efficiency, and number of
eggs recovered between the two methods
applied in the diagnosis of fecal E. paraensei
(Table 1). The method of Hoffman showed the
highest sensitivity when compared to the
method of Kato Katz, indicating positive in all
samples. Regarding the number of eggs
retrieved, the method of Kato Katz showed
lower efficacy (14.35 ± 62.05) when compared
to Hoffman method (165.60 ± 11.22), resulting
in approximately a result 62.53% lower in the
total number of eggs observed (Table 2).
RESULTS
Table 1. Samples and results of fecal eggs counting in hamsters by Kato-Katz and Hoffman techniques.
Parasitological techniques
Hamsters
number Kato-Katz (duplicate) Hoffman (duplicate)
130 47 95 90
12 29 85 82
13 45 105
2
3
4130 75 124
67
92
531 89 85 77
6 12 24 69
7 28 94
8 66 21 91
105
83
98
9 09 37 86 79
10 54 30 94 88
11 43 77 95 75
17 70 97
12
13 69
14 98
57
68
15 72 78 96
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Neotropical Helminthology. Vol. 9, Nº1, jan-jun 2015 Tunholi-Alves et al.
Table 2. Comparison of Techniques Kato-Katz and Hoffman, percentage of positivity and mean number of eggs and
standard error of Echinostoma paraensei in hamsters. X ± SEM Mean ± standard error of the mean.
Positivity (%) X ± SEM
86.66 (13) 62.05 ± 14.35a
Techniques
N
Kato-Katz
15
Hoffman 15 100 (15) 165.60 ± 11.23b
Different letters indicate mean that differ at the 5% level of significance.
identifying the sedimentation technique
spontaneously with sensitivity to the Kato-
Katz. It is noteworthy that studies have shown
higher efficiency of sedimentation techniques,
especially the method of Lutz, for the
diagnosis of trematodes (Palumbo et al., 1976;
Leal, 2003), which was also observed by Silva
& Abboud (200 1) in diagnosing
Platynosomum sp. In this study 100% of
samples were positive method of Hoffman et
al. (1934), agreeing with the results of Holland
(1993) with 85% of positive results for S.
mansoni in relation to the method of Ritchie
(68%), which leads us to consider the
technique of Lutz as the most sensitive in the
recovery of eggs E. paraensei compared with
Kato Katz. Therefore it is recommended
technique for diagnosis in Lutz experimental
infection and epidemiological monitoring
technique, depending on countries considered
endemic, the cultural habit of eating from fish,
crustaceans, amphibians and freshwater
aquatic mollusks raw or undercooked
containing metacercariae and is associated
with low socio-economic conditions, increases
the risk of transmission of human
echinostomiasis, as well as other food-borne
diseases.
This study was supported by Conselho
Nacional para o Desenvolvimento Científico e
Tecnológico (CNPq); Fundação Carlos
Chagas Filho de Amparo à Pesquisa do Estado
do Rio de Janeiro (FAPERJ) and PPGCV-
UFRRJ.
ACKNOWLEDGEMENTS
The data obtained revealed that the Kato-Katz
method showed lower sensitivity (positive)
and specificity when compared to Hoffman
method for the diagnosis of E. paraensei,
which is in agreement with Zaman & Cheong
(1967) to Schistosoma, strengthen and even for
eggs Ascaris and Trichuris. The calculation of
eggs per g by the Kato-Katz method is based on
the assumption that the density of a stool
sample is equal to 1.0, and several factors such
as consistency, preservation solution, parasitic
load and physiology of the host can influence
in the diagnosis of trematodíases (Scott, 1938)
leading to numerous adaptations technique
Kato-Katz (Engels et al., 1993). In this study,
the distribution of eggs of E. paraensei,
followed the pattern of abnormality
demonstrated by dispersion of the values
??around the average and large standard error,
reflecting, thus, a limitation of this technique,
which can be justified by work of Teesdale et
al. (1985). These authors report that a dry stool
sample can provide counts of eggs S. mansoni
seven times greater than a moist sample
obtained from the same host. Watery feces or
very dry and thick fibrous could compromise
the semi-quantitative diagnostic technique
(Feldmeier & Poggensee, 1993). Moreover, in
this study the technique of Lutz showed the
highest sensitivity and efficacy for the
diagnosis of eggs E. paraensei even the
parasite load is low. Similar results were
observed by Santos et al. (2005), in a
comparative study of parasitological methods
for diagnosis of intestinal helminthes
DISCUSSION
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Neotropical Helminthology. Vol. 9, Nº1, jan-jun 2015 Experimental infection by Echinostoma
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