A new laboratorial method for the diagnosis of gastrointestinal parasites in dogs Um novo método laboratorial para diagnóstico de parasitos gastrointestinais em cães

In this study, we aimed to introduce a new technique called TF-Test Modified/Dog for the diagnosis of gastrointestinal parasites in dogs. Fecal samples from 106 dogs were processed by the technique TF-Test Modified/Dog and the techniques of centrifugation-flotation in zinc sulfate, simple-flotation by saturated solution of sodium chloride, direct microscopy exam and TF-Test Conventional. Sensitivity was higher in the TF-Test Modified/Dog (98.41%), followed by flotation in saturated zinc sulfate (77.78%), TF-Test Conventional (73.02%), flotation by saturated sodium chloride (55.55%), and direct microscopy exam (30.16%). The diagnostic efficiency varied from 58.49% to 99.06%, with the highest value also obtained by the new proposed technique. Efficiency level of 99.06% with kappa index 0.979 (almost perfect) was obtained with the TF-Test Modified/Dog. These results represent significant statistical gains (P < 0.05) of 20.63% in sensitivity and 12.27% in efficiency over the best among the other techniques – flotation by saturated zinc sulfate, whose kappa index was 0.738, much lower than that of the TF-Test Modified/Dog. All techniques presented 100% specificity. In this sense, the high sensitivity of the TF-Test Modified/Dog makes it suitable for epidemiological surveys of gastrointestinal parasitosis in dogs, zoonoses control and preventive surveillance programs.


Introduction
The scientific literature reports divergence between the efficiency of different methods for the diagnosis of gastrointestinal parasites in dogs (FOREYT, 1989;TÁPARO et al., 2006;KATAGIRI;OLIVEIRA-SEQUEIRA, 2008).Dogs can as act as sources of infection of gastrointestinal parasites for humans (XIAO et al., 2007;RODIE et al., 2008;BRIDGER;WHITNEY, 2009).In order to evaluate the true extent of the emerging zoonoses, sensitive diagnostic techniques should be widely available (McCARTHY;MOORE, 2000;MEIRELES, 2010).Besides that, improved methods for the detection of parasites in animals and environment would allow for more effective control measures and monitoring strategies.
Among the techniques proposed for fecal examinations in animals, the most common methods for detection of canine gastrointestinal parasitosis are centrifugation-flotation (OLIVEIRA-SEQUEIRA et al., 2002), simple-flotation (SOUSA et al., 2010) and microscopy exam (HENDRIX; ROBINSON, 2006).The technique called Three Fecal Test (TF-Test ® ), here and forth referred to as TF-Test Conventional, was proposed for the diagnosis of intestinal parasites in humans, showing high diagnostic sensitivity (HOSHINO-SHIMIZU et al., 2001;GOMES et al., 2004).The first study using TF-Test Conventional for gastrointestinal parasite detection in animals, specifically in sheep (Ovis aries), was reported by Lumina et al. (2006).The results showed that the TF-Test Conventional could be more sensitive than the modified Gordon & Whitlock technique (UENO; GONÇALVES, 1998) and the techniques of flotation by sodium chloride and direct microscopy exam (GOMES et al., 2006).
In this work, we revisited the methods for dogs by proposing a considerable improvement in the TF-Test Conventional technique with the establishment of TF-Test Modified/Dog, comparing it with the techniques of centrifugation-flotation by saturated solution of zinc sulfate, simple-flotation by saturated solution of sodium chloride and direct microscopy exam.The sensitivity, specificity and efficiency of each technique and their agreement with the Kappa (k) index were determined, classifying them as Poor, Low, Moderate, Substantial and Almost perfect.

Sample collections and laboratory tests
Fecal samples were obtained from 106 dogs kept in kennels in the cities of Botucatu and Bragança Paulista, State of São Paulo, Brazil.A single stool sample was collected from each dog to perform the techniques of centrifugation-flotation by saturated solution of zinc sulfate (FAUST et al., 1938), simple-flotation by saturated solution of sodium chloride (WILLIS, 1921) and direct microscopy exam (KATO; MIURA, 1954).For the TF-Test Conventional (GOMES et al., 2004) and TF-Test Modified/Dog (FALCÃO et al., 2010), fecal samples from each animal were collected using the TF-Test kit in three alternate days.

Statistical analysis
The diagnostic sensitivity, specificity and efficiency of each technique were determined by comparison with reference data.The union of the positive results found by each of the five techniques studied was used as a reasonable approximation of the true positivity (FLEISS, 1981;MacLURE;WILLET, 1987).Similarly, the intersection of the true negative results in all five techniques was also used as reference.The agreement of the techniques with these reference data was calculated by the Kappa (k) index, statistically testing the consistency of k and ranking it as: Poor (0 to 2.0); Low (0.21 to 0.40); Moderate (0.41 to 0.60); Substantial (0.61 to 0.80); and Almost perfect (0.8 to 1.0), according to Gomes et al. (2004).

Results
According to the statistical criteria above, 63 (59.42%) dogs showed positive results by at least one technique, whereas the remaining 43 (40.57%)dogs were considered negative by all five techniques.Among the infected animals, it was possible to verify that 51 (82.26%) dogs had a single type of parasite infection, 8 (12.90%) revealed double infections, and 3 (4.84%)presented triple infections.In the individual evaluation, the positivity was higher in the TF-Test Modified/Dog (58.49%), followed by flotation by saturated zinc sulfate (47.17%),TF-Test Conventional (43,39%), flotation by saturated sodium chloride (33.02%), and direct microscopy exam (17.92%), as shown in Table 1.Single infections were predominantly detected by each of the five techniques, but they varied from 29.03% to 82.26%.
Sixty-three dogs were infected by seven parasite species (Table 2).Infections by three protozoa species were found in 31 (49.21%)dogs and infections by four species of helminths were found in 32 (50.79%) dogs.Among the gastrointestinal parasite species, Giardia spp.and Ancylostoma spp.were detected in a large number of dogs, followed by Entamoeba spp., Trichuris vulpis, Toxocara canis, Isospora spp., and Spirocerca lupi.
The diagnostic performances of the five techniques are shown in Table 3, with the TF-Test Modified/Dog technique presenting the highest sensitivity and efficiency among them.All techniques were able to detect the same 43 dogs as negative, so they presented 100% specificity.
The kappa indexes (k) of the five techniques are shown in Table 4, with statistically significant values (P < 0.05).The TF-Test Modified/Dog was ranked as Almost perfect, whereas the other techniques were ranked from Substantial to Moderate.

Discussion
The positive diagnosis of canine gastrointestinal parasitosis depends on the parasitological technique adopted (GOMES et al., 2006;TÁPARO et al., 2006;KATAGIRI;OLIVEIRA-SEQUEIRA, 2010).In this context, the present study demonstrates interesting aspects of the techniques studied, with significant differences in sensitivity and efficiency.
The proposed technique, TF-Test Modified/Dog, detected 13 infected dogs more than the flotation by the saturated zinc sulfate technique (a gain of 11.32%), 16 infected dogs more than the TF-Test Conventional (a gain of 15.10%), and more than 27 infected dogs with respect to the other techniques (a gain greater than 25.47%).These considerable gains in sensitivity can be justified by the combination of various parasitic enrichment processes, such as: a) collection of fecal material in three alternate days using a preservative solution; b) double filtering of the collected fecal samples; c) two stages of parasitic concentration, centrifugation-sedimentation followed by concentration-flotation; and d) reading of two fecal smears, as recommended in the scientific literature (GARCIA, 2007).The collection of fecal samples in three alternate days using preservative solution considerably favors increased detection of parasitic species, including helminths and protozoa (Table 2).
This happens due to the different biological cycles of each parasite species, some of them with intermittent elimination of parasitic structures.In this case, the suppression of parasitic structures elimination can occur for hours, days, weeks or even months (HOSHINO-SHIMIZU et al., 2001;GOMES et al., 2004;GARCIA, 2007).At the same time, the use of preservative solution preserves the morphology of parasites for a period of thirty days at room temperature (GARCIA, 2007).This is essential for a conclusive diagnosis (GOMES et al., 2004).The double-layered sieves eliminate part of the fecal impurities and the two-stage parasitic concentration -centrifugation-sedimentation followed by concentration-flotation, considerably reduces the remaining impurities.The technique of centrifugation-sedimentation concentrates parasitic structures with density higher than 1.19 g/mL, such as the capsule with eggs of the parasite Dipylidium caninum, for instance.The subsequent flotation-concentration aims at the detection of parasitic structures with density lower than 1.19 g/mL, such as Entamoeba spp., Giardia spp., and Isospora spp.Finally, the TF-Test Modified/Dog adopts two microscope readings.The first aliquot (60µL) examined consists of fecal material processed in the centrifugation-sedimentation step; in the flotation-concentration step, all the sediment from the previous step is used and examined.Helminths and protozoa can be diagnosed in the centrifugationsedimentation aliquot, but after the performance of the flotationconcentration step, protozoa are found in much greater amounts.This way, one can have a good idea of the parasitic infection intensity in dogs.
In the current study, enteroparasitic positivity in dogs varied from 17.92% to 58.49%, depending on the parasitologic technique.These findings emphasize that the choice of a sensitive technique is fundamental to avoid misdiagnosis and, from the viewpoint of public health, implement effective control programs of zoonoses.
We detected 59.42% of infected animals and this positivity is consistent to the one reported by Oliveira-Sequeira et al. (2002) for stray dogs in the State of Sao Paulo Brazil, as determined by the association of three different techniques, such as: sedimentation, simple-flotation and centrifugation-flotation.However, in Chubut, Argentina, the association of these three techniques showed lower prevalence of parasitic infections in stray dogs (SÁNCHEZ-THEVENET et al., 2003).
The techniques studied also detected 82.26% of positive dogs with a single type of infection, 12.90% with double infections, and 4.84% with triple infections.In other localities, such as Neuquén, Argentina, the reported prevalence of multiple parasitic infections was also lower than the single parasitic infection (SORIANO et al., 2009), by the use of two conventional techniques (flotation and sedimentation).Possibly, stray dogs infected by two or more parasite species have lower natural resistance or immunity than those with only one parasite species or without any infection.
Seven parasite species were identified by the five techniques studied: Ancylostoma spp. was detected in 16 (15.09%)dogs by four techniques, except the direct microscopy exam, which detected only half of the cases (7.54%).It is important to emphasize that the adult form of this intestinal helminth, when present in the host, seems to eliminate a great number of eggs, diagnosed even by low sensitive techniques.Other investigators (OLIVEIRA-SEQUEIRA et al., 2002) have also found, in the State of Sao Paulo, no significant difference in the detection of this helminth infection in dogs by the association of three techniques.Nevertheless, in the diagnosis of Giardia spp.and Entamoeba spp., the TF-Test Modified/ Dog was more sensitive than all the other techniques (Table 2), pointing out the influence of the parasitic enrichment procedures and the collection of fecal samples in three alternate days.
Giardia spp.and Ancylostoma spp.were the most prevalent parasites in this study.These parasite species were also detected with moderate and high prevalence in different localities of the State of São Paulo by different authors (GENNARI et al., 2001;OLIVEIRA-SEQUEIRA et al., 2002).Moreover, five out of seven parasite species, Toxocara canis, Ancylostoma spp., Isospora spp., Giardia spp.and Trichuris vulpis, were also detected by the same authors, along with other investigators from Spain (MARTÍNEZ-CARRASCO et al., 2007).
The results obtained with the TF-Test Conventional confirm our previous study in dogs (GOMES et al., 2006), showing that the sensitivity of this technique is lower than the one obtained with flotation by saturated zinc sulfate, which differs from its high sensitivity found for sheep and humans (LUMINA et al., 2006;GOMES et al., 2004).Our findings indicate that dogs eliminate parasitic structures of lower specific densities (less than 1.19 g/mL most of the times) in comparison to sheep and human parasites, resulting in a higher detection of these structures in the flotation using the saturated zinc sulfate technique.However, some species present specific densities higher than 1.19 g/mL, such as Dipylidium caninum egg packets, which do not fluctuate, being detected only by centrifugation-sedimentation.That explains the indication of dual processing in the laboratory routine, such as centrifugation-sedimentation and concentration-flotation.
The highest sensitivity was obtained by the TF-Test Modified/ Dog (98.41%), while the sensitivity of the other techniques varied from 30.16% to 77.78%.This result represents considerable sensitivity gains of 25.39% and 20.63% with respect to the other two most sensitive techniques, TF-Test Conventional and flotation by saturated zinc sulfate, respectively.The diagnostic efficiency varied from 58.49% to 99.06%, with the highest value also obtained by the TF-Test Modified/Dog.All studied techniques presented maximum specificity, since no parasite was found in the same 43 negative dogs.
The agreement of each technique with the reference data revealed similarities and differences between them.The TF-Test Modified/Dog presented a k index ranked as Almost Perfect, being significantly higher than the k indexes of the flotation by saturated zinc sulfate and TF-Test Conventional techniques, both ranked as Substantial.These two techniques, in turn, showed k indexes significantly higher than the flotation by saturated sodium chloride and direct microscopy exam techniques, both at Moderate rank.The TF-Test Modified/Dog has proved to be simple and fast, since it requires less manual handling and less space to be performed.The use of preservative solution prevents the degradation of the parasitic structure, allowing for better visualization.

Table 4 .
Kappa (k)agreement index for different parasitological techniques found in relation to the reference data.

Table 1 .
Positivity and type of infection found in the study of 106 dogs by five parasitologic techniques for fecal examination.

Table 2 .
Parasite species detected in the study of 106 dogs by five parasitologic techniques.

Table 3 .
Diagnostic performance of the five parasitologic techniques in the study of 106 dogs.