Avaliação do SWAB conjuntival para o diagnóstico da Leishmaniose visceral canina por PCR-Hibridização / Evaluation of the conjunctival SWAB for canine visceral Leishmaniasis diagnosis by PCR-Hybridization

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

The visceral leishmaniasis (VL) in Brazil is caused by Leishmania chagasi (L. infantum) and dogs are considered to be the main domestic reservoir. Therefore the correct diagnosis is very important in order to avoid the disease transmission or unnecessary culling of dogs. The aim of this study was to evaluate the conjunctival swab (CS) as a noninvasive sampling method for the canine VL diagnosis by the PCR-hybridization procedure. Firstly, an in vitro test was carried out using cotton swabs seeded with 103 to one cell of L. chagasi. Three DNA purification techniques were tested: phenol-chloroform, Wizard kit and boiling. After that, two groups of 23 seropositive dogs were used. CS samples were obtained from both eyes of each animal. The DNA extraction from CS was performed by the phenol chloroform method in group 1 and by boiling in group 2. In addition, blood was collected from each animal so that 30μL were spotted onto filter paper (FP) and 1.0mL was treated to obtain the buffy coat (BC). The DNA extraction from the BC and FP was accomplished by the same way in both groups using commercial kits. The analysis of all samples was made by PCR associated to the hybridization with 32P labeled DNA probes. The in vitro test with seeded parasites showed positivity to until 25, 10 and one cell for boiling, Wizard kit and phenol chloroform methods respectively. The hybridization step increased the sensitivity until 10 and one cell for the boiling and Wizard respectively. The PCR positivities for the dogs in groups 1 and 2 were respectively: 73.9% and 52.2% (CS), 8,7% and 30.4% (BC), 8.7% and 17.4% (FP). The hybridization step increased the positivities for: 91.3% and 65.2% (CS), 21.7% and 34.8% (BC), 30.4% and 43.5% (FP) respectively. The best frequency of positivity was obtained by the association between CS and the DNA extraction by phenol chloroform. The final result obtained from this treatment was statistically different from the BC and FP data in group 1 (p<0,05). In group 2, the result from CS associated with DNA purification by boiling was distinct only from the BC data (p<0,05). We conclude that the CS associated with the DNA extraction by phenol chloroform is a valuable tool for diagnosis of canine LV and can be recommended for diagnosis of symptomatic dogs.

ASSUNTO(S)

diagnosis infection diseases leishmaniose visceral hibridização leishmaniasis diagnosticos cães hybridization polymerase chain reaction doenças infecciosas pcr dogs parasitologia

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