Comparative study of five techniques for the diagnosis of canine gastrointestinal parasites

Differences in the efficacy of diagnostic techniques employed in the parasitological examination of feces are a limiting factor of this laboratory procedure in the field of Veterinary Parasitology. To verify advances in this type of examination in dogs, we conducted a study using a new technique (TFGII/Dog). Fifty naturally infected dogs were housed in individual stalls, and their feces were evaluated comparatively using this technique and four other conventional techniques. The TFGII/Dog showed high levels of sensitivity and efficiency, surpassing the diagnostic accuracy of the other techniques with a kappa concordance index of 0.739 (Substantial), as opposed to 0.546 (Moderate), 0.485 (Moderate), 0.467 (Moderate), and 0.325 (Fair) of the Spontaneous-Sedimentation, Centrifugal-Flotation in Saturated Zinc Sulfate Solution, Centrifugal-Flotation in Saturated Sugar Solution, and Spontaneous-Flotation in Saturated Sodium Chloride Solution techniques, respectively. The combination of positive results of all techniques comprises eight genera of parasites, with Ancylostoma spp. predominating among helminths, and Cystoisospora spp. among protozoa. The TFGII/Dog technique showed better diagnostic performance, and can therefore be considered an important tool for optimizing the results of laboratory routines and for the control of canine gastrointestinal parasites.


Introduction
Environmental changes caused by humans and their interaction with domestic animals, particularly with dogs, have favored the occurrence of various parasitic diseases with zoonotic potential (BRESCIANI et al., 2008;COELHO et al., 2013). This scenario has led to a high worldwide prevalence of canine gastrointestinal parasites, especially in the tropical regions of underdeveloped and developing countries (CHOMEL, 2008;COELHO et al., 2013).
Numerous studies on dog feces describe discrepancies in the diagnostic efficacy of different parasitological techniques and/or commercial kits (TÁPARO et al., 2006;COELHO et al., 2013). In view of this finding, some researchers recommend the collection of triplicate fecal samples, on alternate days, in combination with the high parasite concentration technique for this same diagnostic modality (GOMES et al., 2004;CARVALHO et al., 2012).
In view of the presented, and unlike Coelho and co-workers (COELHO et al., 2013), and aiming to contribute to scientific and technological advances in the diagnosis of canine gastrointestinal parasites, the current study involved the statistical evaluation of a new parasitological technique (TFGII/Dog), with the implementation of three laboratory processing approaches, based on laboratory principles developed specifically by Falcão et al. for the diagnosis of human intestinal parasites (FALCÃO et al., 2010).

Materials and Methods
Fifty naturally infected dogs used in this study were obtained from the Zoonosis Control Center of the municipality of Andradina, State of São Paulo, Brazil (20.896 South, 51.37944 West, 405m altitude), where they were housed in individual stalls, monitored, watered and fed (dog food). For the TFGII/Dog technique, the feces were identified and analyzed in the laboratory according to the operational protocol described by Falcão et al. in humans (FALCÃO et al., 2011). In addition, fecal samples were collected for laboratory processing by other conventional techniques, such as: spontaneous-flotation using saturated sodium chloride solution with a specific density of 1.20 g/mL (WILLIS, 1921); centrifugal-flotation in a saturated solution of zinc sulfate with a density of 1.18 g/mL (FAUST et al., 1938); centrifugal-flotation in saturated sugar solution with a density 1.18 g/mL (SHEATHER, 1923); and spontaneous-sedimentation (HOFFMAN et al., 1934;LUTZ, 1919). These techniques are referred in this paper as SF-Sodium Chloride, CF-Zinc Sulfate, CF-Sugar and S-Sedimentation, respectively. This study, which is in line with the precepts of the Ethics Committee on Animal Use (CEUA) of UNESP at Jaboticabal, SP, Brazil, was approved on 22 Mar 2011 under Protocol no. 004201/11. The results of this study were applied by means of comparisons of positive data from fecal samples obtained by parasitological techniques. To this end, the following statistical parameters were determined: sensitivity (FLEISS et al., 2003) -detection of true positives animal, specificity (GALEN & GAMBINO, 1975) -detection of true negative animal, and Kappa (k) concordance -agreement degree between qualitative data (positive and negative) obtained by different techniques -and its classification: Almost Perfect (0.81 to 1.00), Substantial (0.61 to 0.80), Moderate (0.41 to 0.60), Fair (0.21 to 0.40) and Slight (0 to 0.20).

Results
In the statistical performance assessment of the five different parasitological techniques, the of TFGII/Dog and S-Sedimentation presented the same diagnostic positivity for 40 dogs (80%), as shown in Table 1. These techniques were followed by the CF-Zinc Sulfate and CF-Sugar techniques, which showed 39 positive dogs (78%), and the SF-Sodium Chloride technique, with 37 positive dogs (74%). However, the type of infection detected, as single, double, triple, quadruple or quintuple, was higher with the use of the parasitological technique of TFGII/Dog.
Eight genera, five of helminths and three of protozoa, were identified by the five parasite detection techniques (Table 2). In the 250 clinical analyses of the feces of the 50 dogs, the TFGII/Dog was the only one able to detect eight different parasitic genera. It is therefore comparable to the Gold Standard (positive results from the combination of all the techniques), since it showed the highest accuracy in the identification of 90 positive analyses. In contrast, based on the same number of clinical analyses (250), seven genera of parasites and 83 positive analyses were identified by the S-Sedimentation; followed by seven genera and 80 positive analyses by the CF-Zinc Sulfate; six genera and 79 positive analyses by the CF-Sugar; and, lastly, six genera of parasites and 69 positive analyses by the SF-Sodium Chloride technique (Table 1).
In accordance with the Gold Standard, among the group of helminths, Ancylostoma spp. predominated in the infections of 35 (70%) dogs ( spp. eggs, the TFGII/Dog proved more accurate than the other conventional techniques. Furthermore, as can be seen in Table 2, despite only one case (dog) of parasitism, Taenia spp. eggs were identified by the TFGII/Dog and by SF-Sodium Chloride, but not by the other techniques, thus putting the former on a par with the Gold Standard. In the diagnosis of Toxocara spp., in turn, the TFGII/Dog, S-Sedimentation and CF-Sugar predominated, producing identical results.
The genus Cryptosporidium spp. was detected in a single animal (2%) by S-Sedimentation, CF-Zinc Sulfate, CF-Sugar and the by TFGII/Dog, which presented the same result as the Gold Standard. Each of these same techniques was also able to identify the parasite Giardia spp. in five (10%) dogs, which also puts them on a par with the Gold Standard (Table 2).
In terms of the diagnostic performance of the techniques, the TFGII/Dog showed 93.75% sensitivity and 94.34% efficiency, compared to 86.46% sensitivity and 87.73% efficiency of S-Sedimentation, 83.33% sensitivity and 84.91% efficiency of CF-Zinc Sulfate, 82.29% sensitivity and 83.96% efficiency of CF-Sugar, and 71.87% sensitivity and 74.53% efficiency of SF-Sodium Chloride (Figure 1).

Discussion
According to Table 1, the total infection rate (positivity and type of infection found in 50 dogs) observed by means of the TFGII/Dog was higher than that identified by the S-Sedimentation, CF-Zinc Sulfate, CF-Sugar and SF-Sodium Chloride techniques. This finding can be explained by the intermittent elimination of the evolutionary forms of each parasitic group, notably enteric protozoa and helminths (CARVALHO et al., 2012;COELHO et al., 2013), indicating that the TFGII/Dog is more efficient, and particularly in cases of multiple parasitism. Two procedures contributed to the high diagnostic efficacy of the TFGII/Dog technique: a) the collection of fecal samples on three alternate days, and their one-time processing in the laboratory (CARVALHO et al., 2012;COELHO et al., 2013); and, b) the application of the three laboratory principles of parasitic concentration, such as, centrifugal-sedimentation, spontaneous-flotation and spontaneous-sedimentation (HOFFMAN et al., 1934;FAUST et al., 1938;GOMES et al., 2004). In these two procedures (a and b), the sample collection and laboratory processing procedures are exclusive to the TFGII/Dog (FALCÃO et al., 2010).   The large number of protozoan cysts and oocysts identified can be explained by the fact that techniques employed in this study take advantage of the selective principles of flotation and sedimentation, which strongly concentrate the structures of these parasitic genera. The fact that we used naturally infected stray dogs that were captured and housed, the low immunity of these animals may have favored the regular elimination of protozoan cysts and oocysts, which, in natural conditions, are expelled intermittently and irregularly in the feces (TÁPARO et al., 2006;BRESCIANI et al., 2008;COELHO et al., 2013).
Although the SF-Sodium Chloride presents a specific flotation principle of parasitic concentration, above all for Ancylostomatidae eggs, the saturated sodium chloride solution (1.20 g/mL) used in this technique causes the lysis of most intestinal protozoa, especially when using a hypertonic medium. Nevertheless, our experience has shown that the genus Cystoisospora spp. is tolerant to this condition (Table 2).
On a par with the Gold Standard, the TFGII/Dog was the only one that was highly efficient in detecting the eight genera of canine gastrointestinal parasites. Thus, this study led to the conclusion that this technique can be widely employed in the qualitative diagnosis of canine gastrointestinal protozoa and helminths, with high performance and is practical, in particular because the kit is completely disposable, thereby reducing the work in the laboratory. It is also cost-effective, with cost equivalent to other conventional techniques and commercial kits (according to the patent applicant -University of Campinas/Inova Innovation Agency). Despite its qualities, the operating protocol of TFGII/Dog must be followed carefully to avoid some sort of limitation to this technique.Lastly, there is no doubt that the gains in sensitivity and efficiency provided by this technique should improve the laboratory diagnosis and control of canine parasitic infections.