Diagnóstico da Leishmaniose Visceral Canina pelas técnicas de imunoistoquímica e PCR em tecidos cutâneos em associação com a RIFI e ELISA-teste

Abstract

ResumoO objetivo deste trabalho foi avaliar as técnicas de imunoistoquímica (IMIQ) e de PCR (Reação em Cadeia da Polimerase) em tecidos cutâneos para o diagnóstico da Leishmaniose Visceral Canina (LVC) e compará-los com os exames parasitológicos em tecidos corados histoquimicamente (hematoxilina-eosina, HE) e com testes sorológicos, como a Reação de Imunofluorescência Indireta (RIFI) e ensaio imunoenzimático (ELISA). Dos 34 cães naturalmente infectados, classificados em assintomáticos, oligossintomáticos e polissintomáticos, foram coletadas amostras de pele sadia ou com lesão para a realização da IMIQ, HE e PCR. Não somente peles lesionadas (56,5%), mas também sadias (31,8%) encontravam-se positivas pela IMIQ, confirmadas posteriormente pela PCR em 97,8% das amostras. No grupo assintomático, 87,5% estavam negativos pelos testes sorológicos, mas positivos em 50% dos casos pela IMIQ e 100% pela PCR. Entre os oligossintomáticos, 100%, 85,7% e 28,6% encontravam-se positivos, respectivamente, pela PCR, sorologia e IMIQ. Os cães polissintomáticos eram 91,7% soropositivos e tinham parasitas na pele. Em geral, a técnica PCR teve maior positividade (100%). A eficiência dos testes variou de acordo com a evolução da doença, demonstrando a necessidade da associação de técnicas, usando-se IMIQ para confirmação da sorologia e a PCR apenas nos casos suspeitos após a IMIQ. Dessa forma, pode-se aumentar os níveis de positividade e contribuir para o controle desta zoonose.Palavras-chave: Leishmania, cães, pele, imunoistoquímica, PCR. AbstractThe purpose of the present study was to evaluate the immunohistochemistry (IMHC) and PCR (Polymerase Chain Reaction) tests for Canine Visceral Leishmaniasis (CVL) diagnosis and compare the results with serological tests such as the indirect fluorescence antibody test (IFAT), ELISA and a parasitological test (microscopic direct examination of the parasite stained with haematoxylin and eosin -HE). For this study, samples of healthy or lesion skin tissues were obtained from 34 CVL naturally infected dogs classified in three groups: asymptomatic, oligosymptomatic and polisymptomatic. Not only lesion (56.5%) but also healthy skins (31.8%) were positives by IMHC and confirmed by PCR in 97.8% of skin samples. In asymptomatic group, 87.5% dogs were negatives by serological tests, but positives by IMHC in 50% and by PCR in 100%. In oligosymptomatic group, 100%, 85.7% and 28.6% of dogs were positives, respectively by PCR, serological and IMHC tests. In addition, 91.7% of polisymptomatic dogs were serum positive and had intact parasites in the skin. In general, PCR showed higher positivity (100%). The efficiency of each test varied with the evolution of the disease. IMHC may be used to confirm the results of the serology and PCR in inconclusive cases after HE and IMHC. The association of techniques proposed in this study may increase the positivity and contributed to the control of this canine disease.

Ability of immunodiagnostic tests to differentiate between dogs naturally infected with Leishmania infantum and Leishmune®-vaccinated dogs

“…These findings are in agreement with de Queiroz et al (2010) who reported that 87.5 % of asymptomatic dogs showed seronegativity in two different tests, while PCR of skin samples revealed 100 % positive results.…”

Use of Parasitological Culture to Detect Leishmania (Leishmania) chagasi in Naturally Infected Dogs

“…This fact may be related to the parasite load in symptomatic animals (Lima et al 2010), thus making it easier to diagnose in this group of animals as proved by Queiroz et al (2010) when using several methodologies for VL diagnosis in dogs with and without clinical symptoms. For Maia et al (2009), the infection difference of diverse organs of the same animal may be associated to the potential parasite load and related with the local immune response of the organ.…”

Molecular detection of Leishmania infantum DNA according to clinical stages of leishmaniasis in dog

“…infection in dogs at several clinical stages and our results showed variation in sensitivity between the primer sets used. The sensitivity of each technique may vary over the course of evolution of the disease and between individuals; therefore, a combination of serological and molecular tests may be necessary to correct the diagnosis (ASSIS et al, 2010;NUNES et al, 2007;QUEIROZ et al, 2010;SILVA et al, 2014). In addition, it has been recommended that the diagnosis of this disease should be based on clinical signs and on the epidemiological characteristics of the region, which may contribute towards correctly identifying truly positive animals (SILVA et al, 2014).…”