(Eh) y metanólico (Em) de Pera distichophylla sobre un aislado de ...
(Eh) y metanólico (Em) de Pera distichophylla sobre un aislado de ... (Eh) y metanólico (Em) de Pera distichophylla sobre un aislado de ...
A. da S. BARBOSA et al. Table 3. Comparison between Ritchie’s modified technique and Sheather’s modified technique for detection of the protozoa Giardia lamblia, Entamoeba histolytica and Cryptosporidium sp. in 64 soil samples Protozoa and helminthes Ritchie’s mod. technique Sheather’s mod. technique MN No.+ No.- No.+ No.- RxS Coccidia oocyst 0 64 2 62 NF Egg of Ascaris sp. 1 63 1 63 1 Nematode egg 1 63 0 64 NF Egg of Trichuris sp. 0 64 2 62 NF 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. Table 4. Comparison between Ritchie’s modified technique, Sheather’s modified technique and ELISA for detection of the protozoa Giardia lamblia, Entamoeba histolytica and Cryptosporidium sp. in 64 soil samples Protozoa ELISA Sheather’s mod. technique/ Ritichie’s mod. Technique MN N+ N- N+ N- ExR / ExS Giardia lamblia 4 60 0 64 NF Cryptosporidium sp. 5 59 0 64 NF Entamoeba histolytica 9 55 0 64 NF 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: McNema Test. only in the detection of Ascaris sp. eggs, which were detected in the same samples. The comparison with other parasites was not possible, because the evolutionary forms were only found using a laboratorial technique (Table 3). In the survey of protozoa G. lamblia and Cryptosporidium sp. in soil samples, the degree of agreement was not calculed, as only ELISA managed to identify the positive samples (Table 4). 94 DISCUSSION Despite the microscopic techniques used in this study presented different principles, an agreement in the detection of cysts of some protozoa could be observed. The Ritchie’s technique modified by Young et al., is based on the centrifugal sedimentation using ethyl acetate, and Sheather’s technique modified by Huber et al., is based on the centrifugal flotation with sucrose solution. These microscopic techniques are widely used in clinical fecal analyses, and, hence, have been evaluated by different authors in fecal examinations. Ritchie’s technique modified by Young et al., presented satisfactory results in children’s feces in the city of Araraquara, State of São Paulo (García et al., 2006) and in feces of patients who lived in slums in the city of Belo Horizonte, State of Minas Gerais (Mello et al., 1989). Sheather’s technique modified by Huber et al., (Huber et al, 2003) has been shown to be highly efficient for the diagnosis of cysts of Giardia sp. and oocysts of Cryptosporidium sp. in calves’ feces. In the soil, the microscopic techniques presented agreement only in the detection of eggs of Ascaris sp., given that most of the positive results were found with Sheather’s modified technique. It should be underscored that, even with gauze filtration with sedimentation, the reading of the slabs for Rev. Ibero-Latinoam. Parasitol. (2012); 71 (1): 90-96
microscopy applying the Ritchie’s modified technique was difficult, as the sediment had become very thick, while in Sheather’s modified technique, owing to the sugar solution with high concentration, the sediment on the microscope slide was clear and easily readable. As an alternative to improve the reading of the slabs in the Ritchie’s modified technique, Carvalho et al., (2002) suggest the dilution of the material obtained with a drop of salt solution for obtainment of clearer fields of reading. Thus, we suggest that Ritchie’s modified technique is not used in the processing of soil samples, as it may lead to false negative results, owing to excess residues in the fields of reading. ELISA showed a larger number of positive samples compared to the microscopic techniques in the survey of G. lamblia, E. histolytica and Cryptosporidium sp. These results are in keeping with the studies performed with fecal samples in the city of Belém, State of Pará, for which ELISA was considered the most sensitive technique for detecting G. lamblia in children’s feces by Machado et al., (2001) and for detecting E. histolytica in feces of dwellers of the Metropolitan Region by Silva et al., (2005). In this study, the agreement between microscopic and ELISA techniques allowed to confirm species of protozoa which could not be identified by microscopy. This fact was substantiated by cysts of Giardia sp. confirmed for G. lamblia and four-nucleus amoeboid cysts confirmed for E. histolytica. The confirmation of species of protozoa through the enzyme immunoassay was possible due to the use of species-specific kits. The degree of agreement between the microscopic techniques and ELISA in soil samples was not ascertained, as protozoa G. lamblia, E. histolytica and Cryptosporidium sp. were detected only in the enzyme immunoassay, and the microscopy did not find any compatible structure. The high sensitivity of the enzyme immunoassay in water and soil samples may be explained by the fact that this technique is capable of capturing surface antigens, given that the integrity of the cyst and oocyst is not necessary. On the other hand, in microscopic techniques, the integrity of the cysts and oocysts of protozoa is essential for the diagnosis. The high efficiency of the enzyme immunoassay is also the result of its ability to detect small amounts of antigens, which stand as low parasite load. This Rev. Ibero-Latinoam. Parasitol. (2012); 71 (1): 90-96 COMPARISON OF TECHNIQUES IN ENVIRONMENTAL SAMPLES may facilitate the detection of antigens in water, since these antigens are usually at low loads, owing to high dilution. Vidal and Catapani (2005) reported that the main disadvantages of enzyme immunoassays are related to the high cost compared to the traditional microscopic techniques. In this study, for each sample, ELISA cost BRL 25.00 in average, while Sheather’s technique modified by Huber et al., cost BRL 0.37, and Ritchie’s technique modified by Young et al., cost BRL 0.55. In addition to this, the enzyme immunoassay, as a specific kit, does not detect other parasites, and the identification of parasite in ELISA does not ensure its feasibility. Comparing the results of ELISA with other immunological techniques is necessary; these techniques include the direct immunofluorescence, which uses monoclonal antibodies with high sensitivity, and is capable of detecting undivided cysts and oocysts. Still, ELISA has the advantage of being less costly than immunofluorescence, is swifter, facilitating concomitant analysis of a number of samples, and is easy to handle. Nonetheless, it has been subject to few tests in environmental samples. Other relevant factors to be considered would be the need for less technology infrastructure, as some kits may be processed by visual reading and do not require a trained operator to identify the evolutionary parasitic forms. The consensus is that the identification of a given parasite must be confirmed by a parasitological laboratory technique. It is recommended, however, the combination of techniques with different principles to increase the probability of detection of evolutionary forms of protozoa and helminths, taking into account the morphological variability they present. However, based on the results found, we suggest the use of immunoenzymatic technique for monitoring the water quality or for investigating environmental contamination, as the mere detection of antigens may indicate risk of infection and, chiefly, the occurrence of fecal source contamination, with the possible presence of other pathogens. REFERENCES 1. CARVALHO FM, FALCÃO AO, ALBUQUERQUE MC, SILVA P, BASTOS OMP, UCHÔA CM. 2002. 95
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A. da S. BARBOSA et al.<br />
Table 3. Comparison between Ritchie’s modified technique and Sheather’s modified technique for <strong>de</strong>tection<br />
of the protozoa Giardia lamblia, Entamoeba histolytica and Cryptosporidium sp. in 64 soil samples<br />
Protozoa and helminthes<br />
Ritchie’s mod. technique Sheather’s mod. technique MN<br />
No.+ No.- No.+ No.- RxS<br />
Coccidia oocyst 0 64 2 62 NF<br />
Egg of Ascaris sp. 1 63 1 63 1<br />
Nemato<strong>de</strong> egg 1 63 0 64 NF<br />
Egg of Trichuris sp. 0 64 2 62 NF<br />
No.+ Number of positive samples; No.-: Number of negative samples; E: ELISA; R: Ritchie’s technique modified<br />
by Yo<strong>un</strong>g et al., S: Sheather’s technique modified by Huber et al.; NF: Comparison was not possible; MN: McNemar<br />
Test.<br />
Table 4. Comparison between Ritchie’s modified technique, Sheather’s modified technique and ELISA for<br />
<strong>de</strong>tection of the protozoa Giardia lamblia, Entamoeba histolytica and Cryptosporidium sp. in 64 soil samples<br />
Protozoa ELISA Sheather’s mod. technique/<br />
Ritichie’s mod. Technique<br />
MN<br />
N+ N- N+ N- ExR / ExS<br />
Giardia lamblia 4 60 0 64 NF<br />
Cryptosporidium sp. 5 59 0 64 NF<br />
Entamoeba histolytica 9 55 0 64 NF<br />
No.+ Number of positive samples; No.-: Number of negative samples; E: ELISA; R: Ritchie’s technique modified<br />
by Yo<strong>un</strong>g et al., S: Sheather’s technique modified by Huber et al., NF: Comparison was not possible; MN: McNema<br />
Test.<br />
only in the <strong>de</strong>tection of Ascaris sp. eggs, which<br />
were <strong>de</strong>tected in the same samples. The comparison<br />
with other parasites was not possible, because<br />
the evolutionary forms were only fo<strong>un</strong>d using a<br />
laboratorial technique (Table 3).<br />
In the survey of protozoa G. lamblia and<br />
Cryptosporidium sp. in soil samples, the <strong>de</strong>gree<br />
of agreement was not calculed, as only ELISA<br />
managed to i<strong>de</strong>ntify the positive samples (Table 4).<br />
94<br />
DISCUSSION<br />
Despite the microscopic techniques used in this<br />
study presented different principles, an agreement<br />
in the <strong>de</strong>tection of cysts of some protozoa could<br />
be observed. The Ritchie’s technique modified by<br />
Yo<strong>un</strong>g et al., is based on the centrifugal sedimentation<br />
using ethyl acetate, and Sheather’s technique<br />
modified by Huber et al., is based on the centrifugal<br />
flotation with sucrose solution.<br />
These microscopic techniques are wi<strong>de</strong>ly used<br />
in clinical fecal analyses, and, hence, have been<br />
evaluated by different authors in fecal examinations.<br />
Ritchie’s technique modified by Yo<strong>un</strong>g et al.,<br />
presented satisfactory results in children’s feces in<br />
the city of Araraquara, State of São Paulo (García et<br />
al., 2006) and in feces of patients who lived in slums<br />
in the city of Belo Horizonte, State of Minas Gerais<br />
(Mello et al., 1989). Sheather’s technique modified<br />
by Huber et al., (Huber et al, 2003) has been shown<br />
to be highly efficient for the diagnosis of cysts of<br />
Giardia sp. and oocysts of Cryptosporidium sp. in<br />
calves’ feces.<br />
In the soil, the microscopic techniques presented<br />
agreement only in the <strong>de</strong>tection of eggs of<br />
Ascaris sp., given that most of the positive results<br />
were fo<strong>un</strong>d with Sheather’s modified technique. It<br />
should be <strong>un</strong><strong>de</strong>rscored that, even with gauze filtration<br />
with sedimentation, the reading of the slabs for<br />
Rev. Ibero-Latinoam. Parasitol. (2012); 71 (1): 90-96
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