Técnica da imunoperoxidase utilizando um soro hiperimune anti-Leishmania (L.) chagasi no diagnóstico da leishmaniose tegumentar americana confirmada por cultura

The present study reports the production of the rabbit anti-Leishmania (L.) chagasi hyperimmune serum, the standardization of the immunohistochemistry (IHC) technique and the evaluation of its employment in cutaneous leishmaniasis (CL) lesions diagnosed by Leishmania sp. culture isolation. Thirty fragments of active CL lesions were examined as well as 10 fragments of cutaneous mycosis lesions as control group. IHC proved more sensitive in detecting amastigotes than conventional hematoxylin-eosin (HE) stained slides: the former was positive in 24 (80%) biopsies whereas the latter, in 16 (53%) (p = 0.028). The reaction stained different fungus species causing cutaneous mycosis. Besides, positive reaction was noticed in mononuclear and endothelial cells. Nevertheless, this finding was present in the control group biopsies. It is concluded that IHC showed good sensitivity in detecting amastigotes.O presente estudo relata a produção do soro policlonal de coelho anti-Leishmania (L.) chagasi, a padronização da técnica de imunohistoquímica (IHQ) e sua aplicação em lesões de leishmaniose cutânea (LC) diagnosticadas por isolamento de Leishmania sp. em cultura. Foram examinados 30 fragmentos de lesões ativas de LC e 10 fragmentos de lesões de etiologia fúngica, utilizados como grupo controle. A IHQ mostrou-se mais sensível na detecção de amastigotas que a coloração em hematoxilina-eosina (HE), sendo positiva em 24 fragmentos de LC (80%) e ao passo que a HE foi positiva em 16 (53%) (p = 0,028). A IHQ também marcou diferentes espécies de fungos causadoras de micoses cutâneas. Adicionalmente, verificou-se positividade no citoplasma de células mononucleares e células endoteliais. Entretanto, esse achado esteve presente no grupo controle. Conclui-se que o método de IHQ apresentou boa sensibilidade na detecção de formas amastigotas

A utilização do ELISA empregando antígenos homólogos e heterólogos para a detecção de IgG e subclasses (IgG1 e IgG2) no diagnóstico de Leishmaniose visceral canina

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.A imunofluorescência indireta é o método recomendado para o diagnóstico de leishmaniose visceral em cães, entretanto, a acurácia dessa técnica é baixa e seu uso em grande escala é limitado. Uma vez que o ELISA não apresenta essas limitações, essa técnica poderia ser uma opção para a detecção de IgG ou subclasses IgG1 e IgG2 específicas. A ehrlichiose canina é um importante diagnóstico diferencial de Leishmaniose Visceral Americana (LVA). O presente estudo comparou o ELISA usando antígenos de Leishmania chagasi e Leishmania braziliensis para a detecção de IgG e subclasses anti-Leishmania em amostras de soro de 37 cães naturalmente infectados com L. chagasi (LVA) e em amostras de quatro cães co-infectados (CI). A ocorrência de reatividade cruzada foi investigada em amostras de soro controle de 17 animais saudáveis (HC) e 35 de infectados por Ehrlichia canis (EC). A média de densidade óptica obtida para a detecção de IgG foi significantemente maior quando o antígeno de L. chagasi foi usado e também mais elevada no subgrupo LVs (sintomático) quando comparado ao subgrupo LVa (assintomático). A correlação entre IgG e IgG1 foi baixa. O presente resultado sugere que ELISA IgG empregando antígeno homólogo, produz os melhores resultados, permitindo o diagnóstico de infecção assintomática por L. chagasi e a discriminação entre casos de LVA e ehrlichiose em cães

Use of elisa employing homologous and heterologous antigens for the detection of IgG and subclasses (IgG1 and IgG2) in the diagnosis of Canine visceral leishmaniasis

Submitted by Repositório Arca ([email protected]) on 2019-04-24T16:40:53Z No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Approved for entry into archive by Janaína Nascimento ([email protected]) on 2019-11-19T14:02:14Z (GMT) No. of bitstreams: 2 ve_Ribeiro_Flávio_etal_INI_2011.pdf: 961127 bytes, checksum: b5f638cb76382681343ff10faf62fbb4 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-11-19T14:02:15Z (GMT). No. of bitstreams: 2 ve_Ribeiro_Flávio_etal_INI_2011.pdf: 961127 bytes, checksum: b5f638cb76382681343ff10faf62fbb4 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Laboratório de Vigilância em Leishmanioses. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Laboratório de Vigilância em Leishmanioses. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Laboratório de Vigilância em Leishmanioses. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Laboratório de Pesquisa Clínica em Dermatozoonoses em Animais Domésticos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Laboratório de Vigilância em Leishmanioses. Rio de Janeiro, RJ, Brasil.Instituto Municipal de Medicina Veterinária Jorge Vaitsman. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Laboratório de Pesquisa Clínica em Dermatozoonoses em Animais Domésticos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Laboratório de Vigilância em Leishmanioses. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Laboratório de Vigilância em Leishmanioses. Rio de Janeiro, RJ, Brasil.Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs

(Upper panel) Parasite enumeration during pregnancy and postpartum.

<p>Amastigotes present in the lesions were enumerated by in situ immunohistochemistry in active cutaneous lesions of both pregnant patients (PP1, PP2) before treatment, in the eighth month of pregnancy (gray bars) and 2–6 months after birth (black bars). Amastigote numbers present in lesions of nonpregnant ATL patients (average of three patients) before treatment are shown as comparison. The results are expressed as number of parasites per mm², using a grid-scale consisting of 20×20 subdivisions in an area of 10 mm²×10 mm², adapted to slides as previously described <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002472#pntd.0002472-Morgado2" target="_blank">[24]</a>. The results shown are median ± SEM. n.d. = not detectable; (Lower panel) Serum arginase activity. Arginase activity (U/mL) was determined in sera obtained from peripheral blood of pregnant ATL patients collected at 3, 5, and 9 months of pregnancy (black bar PP1; grey bar PP2) as well as four age-matched nonpregnant ATL patients (hatched bars). The results shown are median ± SEM.</p

Determination of IFN-γ and IL-10–producing cells in the peripheral blood during pregnancy and postpartum.

<p>(A) The number of IFN-γ and (B) of IL-10–producing cells in peripheral blood was determined at 8 months of pregnancy and 2–6 months after birth in both pregnant ATL patients by ELISPOT assay and compared to those present in nonpregnant ATL patients before specific treatment and in healthy volunteers. Spontaneous release (black bars) as well as antigen-specific responses to restimulation with <i>Leishmania</i>-antigen in vitro (grey bars) are shown. The results from antigen-stimulated PBMC were expressed as the difference of the number of spots/10,000 PBMC in the antigen-stimulated wells minus the mean number of spots in control wells (with medium alone = spontaneous release) from the same donor. The results shown are median ± SEM.</p