Comparative study of in situ hybridization, immunohistochemistry and parasitological culture for the diagnosis of canine leishmaniosis

Abstract

BackgroundThe establishment of an accurate diagnostic protocol for canine visceral leishmaniosis (CanL) is a significant laboratory challenge and the lack of a reliable reference standard is one of the major problems. The aim of this study was to compare in situ hybridization (ISH), immunohistochemistry (IHC) and parasitological culture (PC) for detection of L. infantum in skin, spleen, lymph node and bone marrow of clinically healthy and sick seropositive dogs.FindingsThe study included 65 dogs positive with both DPP® and ELISA for anti-Leishmania antibodies. In situ hybridization of spleen or lymph node had the highest positivity rates of L. infantum detection. The total positivity rates for IHC, ISH and PC were 70 %, 68.1 % and 65.8 %, respectively. When combining techniques, the positivity rates were 81.5 % in the spleen, 79.0 % in lymph nodes, 59.0 % in bone marrow and 52.3 % in the skin. The highest percentage of infected dogs (87.7 %) was detected by using lymph node samples. When examining only skin, positivity was significantly higher in sick dogs than in the clinically healthy dogs. Infection with L. infantum was confirmed in 95.8 % of sick dogs and in 82.4 % of healthy dogs.ConclusionsConsidering the advantages of accurately diagnosing different Leishmania species and of being more sensitive than PC, ISH should be considered as reference standard test for the diagnosis of CanL. Spleen and lymph node are the most suitable tissues to confirm infection with L. infantum in seropositive dogs. The testing of only skin from clinically healthy dogs may result in a high percentage of false negative results.

Comparison between Colorimetric In Situ Hybridization, Histopathology, and Immunohistochemistry for the Diagnosis of New World Cutaneous Leishmaniasis in Human Skin Samples

“…This lower sensitivity of in situ hybridization compared with PCR is due to the fact that there is no amplification of parasite DNA in this technique as in PCR, but rather a detection of nucleic acids in the sample [11,14]. Nonetheless, the main advantages of in situ hybridization compared with PCR are the abilities to diagnose active infections and allow for the correlation between the presence of the parasite and the lesion [35]. In addition, PCR may have inaccuracies in sensitivity, especially in FFPE tissues [8,9], and a combination with a parasitological or histological technique is recommended to increase accuracy in the diagnosis of human NWCL [3].…”

Frequency of detection and load of amastigotes in the pancreas of Leishmania infantum-seropositive dogs: clinical signs and histological changes

“…amastigotes in the pancreas observed in the present study was slightly lower than the frequency of 22.6% reported by Guerra et al [ 15 ] using IHC. However, this frequency was much lower than that obtained by IHC in other organs of dogs that tested seropositive for anti- L. infantum antibodies by TR DPP® and ELISA, such as spleen (78.79 to 81.5%), bone marrow (64.6%), liver (73.6%), lymph node (76.9 to 81.5%), skin (45.6 to 65.4%), and genital tract (85.0 to 90.0%) [ 15 , 26 , 31 ]. Unlike organs of the mononuclear phagocyte system, skin, and genital tract, these results suggest the absence of tropism of L. infantum for the pancreas [ 15 , 26 , 31 ].…”

“…However, this frequency was much lower than that obtained by IHC in other organs of dogs that tested seropositive for anti- L. infantum antibodies by TR DPP® and ELISA, such as spleen (78.79 to 81.5%), bone marrow (64.6%), liver (73.6%), lymph node (76.9 to 81.5%), skin (45.6 to 65.4%), and genital tract (85.0 to 90.0%) [ 15 , 26 , 31 ]. Unlike organs of the mononuclear phagocyte system, skin, and genital tract, these results suggest the absence of tropism of L. infantum for the pancreas [ 15 , 26 , 31 ]. On the other hand, the frequency of pancreatic infection might be higher than that found in this study since the sensitivity of IHC for the diagnosis of L. infantum infection in tissues of dogs is lower than that of the polymerase chain reaction [ 32 , 33 ].…”

Detecção de antígenos circulantes como abordagem diagnóstica em leishmaniose visceral.

“…No Brasil, a LV é considerada uma doença endêmica e encontra-se em franca expansão geográfica, o que caracteriza sua urbanização e uma mudança em seu perfil de transmissão, antes restrito a áreas rurais. Como consequência deste processo, o relato de surtos em centros urbanos é cada vez mais frequente, como descrito em estudos nos estados de Pernambuco, São Paulo, Maranhão e Rio de Janeiro (Dantas-Torres, Brandão-Filho, 2006;Silva et al, 2008;D'Andrea et al, 2015;Furtado et al, 2015;).…”