Estudo comparativo dos métodos diagnósticos para Leishmaniose Visceral em cães oriundos de Ilha Solteira, SP

Assis, Juliana de; Queiroz, Nina Marí Gual Pimenta de; Silveira, Rita de Cássia Viveiros da; Nunes, Cáris Maroni; Oliveira, Trícia Maria Ferreira de Sousa; Noronha, Antonio Carlos Faconti de; Neves, Maria Francisca; Machado, Rosangela Zacarias; Buzetti, Wilma Aparecida Starke

doi:10.4322/rbpv.01901004

Cited by 18 publications

(18 citation statements)

References 23 publications

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“…According to the tests, the positivism rates for CVL were the following: 71.6% (48/67), 65.7% (44/67), 65.7% (44/67) and 55.2% (37/67) by ELISA-SE, ELISA-rk39, IFAT and RDT-rK39, respectively. Furthermore, the results showed that the serum prevalence rates were higher in symptomatic rather than in asymptomatic dogs (Table 1) in accordance to the results of Assis et al (2010) and Queiroz et al (2010), who compared different diagnostic tests in a group of CVL positive dogs.…”

Section: Resultssupporting

confidence: 83%

“…The Kappa indexes of agreement (Table 2) between both serological methods (ELISA-SE × IFAT) were considered good in the symptomatic (87.3%, k= 0.6867) and excellent in the asymptomatic group (91.7%, k= 0.8333). Similarly, Assis et al (2010) observed also a good concordance between ELISA-SE and IFAT, indicating that either ELISA or IFAT can be recommended for CVL diagnosis during epidemiological surveys.…”

Section: Resultsmentioning

confidence: 65%

“…The PA is a very specific method for CVL diagnosis, since it detects the parasites in the tissues, including biopsies or/and aspirates of bone marrow, lymph nodes, spleen, liver or skin (ALVAR et al, 2004;MOREIRA et al, 2007;TASCA et al, 2009;ASSIS et al, 2010;QUEIROZ et al, 2010). However, the sensitivity of this method is low, depending on biologic material, parasitic load and animal's clinical status (GENARO, 1993).…”

Section: Resultsmentioning

confidence: 99%

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Comparative evaluation of several methods for Canine Visceral Leishmaniasis diagnosis

Silva

Starke-Buzetti

Alves-Martin

et al. 2014

Rev. Bras. Parasitol. Vet.

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The aim of the present study was to evaluate the serological methods using ELISA with recombinant-rK39 (ELISA-rK-39) and soluble extract-SE (ELISA-SE) antigens, the indirect fluorescence antibody test (IFAT) in comparison to an immunochromatography rapid diagnostic test (RDT-rK39) and with a direct parasitological exam (PA) for Canine Visceral Leishmaniasis (CVL) diagnosis. The results showed that 89% (60/67) of the dogs were positive for at least one serological diagnostic test. ELISA-SE was the test that detected anti-Leishmania antibodies in the serum of the highest number of dogs (71.6%) followed by ELISA-rK39 (65.7%), IFAT (65.7%) and RDT-rK39 (55.2%). PA detected the lowest numbers (40.3%) of positive dogs. In relation to the total of examined dogs, the Kappa indexes (p ≤ 0.05) showed a good agreement between ELISA-SE and IFAT (88.1%; k = 0.7237), and it was also observed in the comparison of RDT-rK39 with ELISA-SE (83.6%, k= 0.6561), IFAT (83.5%, k= 0.6605) and PA (85.0%, k= 0.7074). A bad agreement was detected in any association of ELISA-rk39 with the other tests in either symptomatic or asymptomatic animals. ELISA as well as RDT using recombinant antigenic protein (rK39) were the methods that detected the lowest prevalence rates (33.3%) of CVL in asymptomatic dogs. In conclusion, only one test does not adequately identify dogs with CVL and it is necessary the association of two or more diagnostic tests. Because of the good agreement indexes of RDT-rK39 when evaluated with ELISA-SE, IFAT and PA it was suggested as a complementary method to be used in association with either ELISA-SE or IFAT, particularly in the symptomatic dogs. Furthermore, new studies are recommended in order to improve the sensitivity of tests mainly for asymptomatic dogs.Keywords: CVL, diagnosis, dogs, Leishmania infantum, rK39, RDT. ResumoO objetivo do presente estudo foi avaliar os métodos sorológicos usando ELISA (Ensaio Imunoenzimático Indireto) com o antígeno recombinante rK39 (ELISA-rK39) e o antígeno extrato solúvel bruto (ELISA-ES) e a RIFI (Reação de Imunofluorescência Indireta) em comparação com o método imunocromatográfico rápido (RDT-rK39) e o parasitológico direto (PA), para o diagnóstico da Leishmaniose Visceral Canina (LVC) em cães de Ilha Solteira, São Paulo, Brasil. Os resultados mostraram que 89% (60/67) dos cães foram positivos por pelo menos um teste diagnóstico sorológico (RIFI, ELISA-ES, ELISA-rk39 ou RDT-rK39) e somente 40,3% (27/67) foram positivos pelo PA. O ELISA-ES foi o teste que detectou anticorpos anti-Leishmania em maior número de cães (71,6%) seguido por ELISArK39, RIFI (65,7%) e por 2%). No total de cães analisados (assintomáticos e sintomáticos), o índice Kappa de concordância (p ≤ 0,05) foi considerado de boa concordância entre ELISA-ES e IFAT (88,1%; k= 0,7237) e entre RDT-rK39 com ELISA-ES (83,6%, k= 0,6561), RIFI (83,5%, k= 0,6605) e PA (85,0%, k= 0,7074). O índice de concordância ruim foi observado em qualquer associação de ELISA-rk39 com todos os outros testes nos animais sintomáticos ...

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Section: Resultssupporting

confidence: 83%

Section: Resultsmentioning

confidence: 65%

Section: Resultsmentioning

confidence: 99%

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Comparative evaluation of several methods for Canine Visceral Leishmaniasis diagnosis

Silva

Starke-Buzetti

Alves-Martin

et al. 2014

Rev. Bras. Parasitol. Vet.

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“…Then, the serological tests of these animals were repeated in our laboratory using an indirect ELISA test and IFAT (indirect fluorescent antibody test) according to Oliveira et al (2008), and molecular analysis of the blood were also performed by PCR (polymerase chain reaction) according to standard protocol used by Assis et al (2010). All these tests were again positive for CVL.…”

Section: Cvl Infected Dogs (G1 and G2 Group)mentioning

confidence: 99%

Correlation study and histopathological description of intestinal alterations in dogs infected with Leishmania infantum

Silva

Neves

Queiroz

et al. 2016

Rev. Bras. Parasitol. Vet.

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The aim of this work was a correlation study and histopathological description of alterations associated with the presence of Leishmania infantum amastigote in the intestinal wall of dogs infected with canine visceral leishmaniasis (CVL). Three groups were used: G1 (n = 8), comprising naturally infected dogs with CVL with amastigotes of L. infantum in the small and large intestines; G2 (n = 9), infected dogs with CVL, without intestinal amastigotes; and G3 (n = 3), uninfected dogs. Histochemistry and immunohistochemistry methods were used for histopathology and amastigotes identification. 47.1% (8/17) of dogs from G1 group had amastigotes in the mucosa, submucosa and muscle layers of the small and large intestines and it was observed a prominent inflammatory reaction characterized by chronic infiltration of mononuclear cells: macrophages, lymphocytes and plasma cells. Comparison between the groups showed only a significant difference in relation to mucosal microscopic structural alterations in dogs from G1 in relation to G2 and G3. Parasite burden showed significant correlations with the microscopic alterations and clinical status of dogs in G1. By the conclusion, the inflammatory reactions caused by the parasites in the intestines might have contributed towards alterations in digestive processes, worsening the dogs' clinical status of CVL.Keywords: Dog, histology, immunohistochemistry, intestine, leishmaniasis, amastigotes. ResumoO objetivo foi realizar um estudo de correlação e descrição histopatológica das lesões associadas à presença de amastigotas de Leishmania infantum na parede intestinal de cães infectados com leishmaniose visceral canina (LVC). Os cães foram subdivididos em três grupos: G1 (n = 8) cães naturalmente infectados com LVC e com amastigotas de L. infantum no intestino; G2 (n = 9) com LVC, mas sem o parasitismo intestinal; e G3 (n = 3) cães não infectados. Métodos histoquímicos e imunoistoquímicos foram utilizados para a histopatologia e a identificação das amastigotas, respectivamente. 47,1% (8/17) dos cães infectados (grupo G1) apresentavam formas amastigotas na mucosa, submucosa e camada muscular do intestino delgado e grosso, destacando-se uma reação inflamatória caracterizada por infiltrado crônico de células mononucleares; macrófagos, linfócitos e plasmócitos. Observou-se uma diferença significativa somente com relação às alterações estruturais microscópicas intestinais nos cães do G1 quando comparadas com G2 e G3. A intensidade parasitária intestinal teve correlação significativa com as alterações microscópicas e os sinais clínicos dos cães do G1. Concluiu-se que as amastigotas de L. infantum por causarem reações inflamatórias na parede intestinal dos cães podem ter contribuído para as alterações dos processos digestórios, agravando ainda mais o quadro clínico dos animais.Palavras-chave: Cão, histologia, imunoistoquímica, intestino, leishmaniose, amastigotas.

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“…Molecular tools for the diagnosis of Leishmania infections are routinely employed for humans, vectors, and wild and domestic mammalian samples [6,13,16–17]. This intracellular parasite is heterogeneously distributed in mammal tissues and is usually described in low parasite loads, at least in wild mammals [2].…”

Section: Discussionmentioning

confidence: 99%

Multiplex PCR as a tool for the diagnosis of Leishmania spp. kDNA and the gapdh housekeeping gene of mammal hosts

et al. 2017

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BackgroundThe PCR assays usually employed for Leishmania diagnosis does not simultaneously detect a constitutive gene that would certify the viability of the DNA sample. We present a multiplex PCR approach for the simultaneous diagnosis of the Leishmania sp. kDNA fragment and a catalytic domain segment of a conserved region of the mammalian gapdh gene.MethodologyThe proposed multiplex protocol was designed through in silico PCR. The annealing temperature, concentration of primer pairs, number of cycles, distinct polymerase enzymes and premix kit were defined to achieve an optimal reaction. The DNA detection sensitivity was tested with different concentrations of known L. tropica DNA, and the reproducibility of the assay was confirmed using samples from 106 wild mammals that were previously subject to Leishmania sp. kDNA analysis through singleplex reactions.Principal findingsThe following optimal conditions were established: 95°C for 1 min followed by 30 cycles of 95°C for 30 s, 61°C for 30 s, and 72°C for 30 s and a final extension at 72°C for 1 min. The multiplex PCR system was capable of detecting 0.1 ng of L. tropica diluted in 100 ng of mammalian DNA. Of 51 kDNA samples that were previously found to be positive, 45 (88.2%) were positive for both targets, two were positive only for kDNA and four were negative for both. Of 55 kDNA samples that were previously identified as negative, 38 (69.1%) were positive for gapdh whereas the other 17 were negative for both targets.Conclusions/SignificanceThe proposed multiplex PCR system allows the simultaneous detection of the gapdh gene and Leishmania sp. kDNA in tissue samples derived from distinct wild mammal species. The amplification of the gapdh mammalian gene in the same reaction ensures the quality and viability of the DNA in the sample, eliminating the possibility of false-negative results that would impair an accurate description of the infection rates in a given population.

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Estudo comparativo dos métodos diagnósticos para Leishmaniose Visceral em cães oriundos de Ilha Solteira, SP

Cited by 18 publications

References 23 publications

Comparative evaluation of several methods for Canine Visceral Leishmaniasis diagnosis

Comparative evaluation of several methods for Canine Visceral Leishmaniasis diagnosis

Correlation study and histopathological description of intestinal alterations in dogs infected with Leishmania infantum

Multiplex PCR as a tool for the diagnosis of Leishmania spp. kDNA and the gapdh housekeeping gene of mammal hosts

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