Acurácia e confiabilidade do ensaio imunoenzimático produzido por Bio-Manguinhos/FIOCRUZ para o diagnóstico da leishmaniose visceral canina / Accuracy and reliability of Bio-Manguinhos/FIOCRUZ produced by enzyme immunoassay for the diagnosis of canine visceral leishmaniasis

Em decorrência da importância que o cão doméstico exerce frente à manutenção do ciclo de transmissão da leishmaniose visceral (LV), uma das estratégias de controle da doença é a eliminação de cães soropositivos. No entanto, até o início deste trabalho, ainda não havia sido realizado um estudo de acurácia diagnóstica com tamanho amostral expressivo do ensaio imunoenzimático (EIE) disponível no Brasil para diagnóstico de leishmaniose visceral canina (LVC). Neste estudo foi avaliada a acurácia, reprodutibilidade e a validação do EIE para o diagnóstico da leishmaniose visceral canina (EIE-LVC) produzido pelo Instituto de Tecnologia em Imunobiológicos (Bio-Manguinhos). Foram incluídos 802 cães de áreas endêmicas captados por vigilância ativa e em Centros de Controle de Zoonoses (CCZ). Para o estudo de confiabilidade toda a amostra foi analisada de forma mascarada e em duplicata e 167 amostras foram repetidas também em outro laboratório (CCZ-Belo Horizonte). Foram considerados cães com LVC os que apresentaram diagnóstico parasitológico positivo com isolamento de Leishmania (Leishmania) chagasi ou imunofluorescência indireta (IFI) e reação em cadeia da polimerase (PCR) positivos. A confiabilidade das observações intraobservador foi excelente, com coeficiente de correlação intraclasse (CCI) de 0,98 (Intervalo de Confiança - IC 95 por cento 0,98;0,99) para toda amostra e excelentes coeficientes de correlação de concordância de Lin...

Para a confiabilidade entre observadores, o resultado também foi excelente. Os parâmetros de acurácia calculados para as duas aferições realizadas com toda amostra (N=802) indicaram sensibilidade de 89,9 por cento (IC 95 por cento 86,5;92,7) e 87,6 por cento (IC 95 por cento 83,9;90,7) para a 1ª e 2ª aferições, respectivamente, e especificidade de 89,4 por cento (IC 95 por cento 86,0;92,2) e 91,6 por cento (IC 95 por cento 88,5;94,1). A acurácia foi alta independente da condição clínica do cão com áreas sobre a curva ROC (Receiver Operating Characteristic) grandes variando de 0,94 a 0,92, e como esperado, os valores preditivos positivos aumentaram com a piora da condição clínica do cão. O desempenho do teste EIELVC o credencia para o diagnóstico em população de cães suspeitos de LVC...

[Report on an autochthonous case of canine visceral leishmaniasis in the southern zone of the municipality of Rio de Janeiro]. / Relato de caso autóctone de leishmaniose visceral canina na zona sul do município do Rio de Janeiro.

Brazil is facing expansion and urbanization of American visceral leishmaniasis, with human and canine cases in several large-sized cities. This report describes an autochthonous case of canine visceral leishmaniasis in a nonendemic area in the municipality of Rio de Janeiro.

Relato de caso autóctone de leishmaniose visceral canina na zona sul do município do Rio de Janeiro / Report on an autochthonous case of canine visceral leishmaniasis in the southern zone of the municipality of Rio de Janeiro

O Brasil enfrenta uma expansão e urbanização da leishmaniose visceral americana com casos humanos e caninos em várias cidades de grande porte. O presente relato descreve um caso de leishmaniose visceral canina autóctone em uma área não endêmica no município de Rio de Janeiro.

Brazil is facing expansion and urbanization of American visceral leishmaniasis, with human and canine cases in several large-sized cities. This report describes an autochthonous case of canine visceral leishmaniasis in a nonendemic area in the municipality of Rio de Janeiro.

Viability study of a multiplex diagnostic platform for Chagas disease.

A new multiplex assay platform was evaluated to detect Trypanosoma cruzi infection using the recombinant antigens CRA, FRA, CRAFRA fusion and parasite lysate. The antigens presented different sensitivity and specificity in a singleplex test when compared to a serial dilution of two pools comprising 10 positive serum samples and one pool of 10 negative samples. The recombinant protein CRA presented lower sensitivity (55%) in contrast to the 100% specificity and sensitivity of FRA, CRAFRA and T. cruzi lysate. These antigens also showed good results in a duplex test and the duplex test with CRAFRA/T. cruzi lysate showed better performance with 100% specificity and sensitivity, as well as a lower cut-off value in comparison to the other duplex test, FRA/T. cruzi lysate. Hence, when the antigens were used in duplex format, both tests showed decreased cut-off values and no interference between different bead sets, resulting in increasing sensitivity and specificity. The results of these multiplex tests show that they could be an alternative to singleplex detection for Chagas disease, and also indicate the necessity of using multiplex diagnostic tools to increase the sensitivity and specificity for diagnostic tests. Emerging data from the T. cruzi genome and from its ORFeome project will also allow the identification of new antigens for this disease detection application.

Viability study of a multiplex diagnostic platform for Chagas disease

A new multiplex assay platform was evaluated to detect Trypanosoma cruzi infection using the recombinant antigensCRA, FRA, CRAFRA fusion and parasite lysate. The antigens presented different sensitivity and specificity in a singleplex test when compared to a serial dilution of two pools comprising 10 positive serum samples and one pool of 10 negative samples. The recombinant protein CRA presented lower sensitivity (55%) in contrast to the 100% specificity and sensitivity of FRA, CRAFRA and T. cruzi lysate. These antigens also showed good results in a duplex test and the duplex test with CRAFRA/T. cruzi lysate showed better performance with 100% specificity and sensitivity, as well as a lower cut-off value in comparison to the other duplex test, FRA/T. cruzi lysate. Hence, when the antigens were used in duplex format, both tests showed decreased cut-off values and no interference between different bead sets, resulting in increasing sensitivity and specificity. The results of these multiplex tests show that they could be an alternative to singleplex detection for Chagas disease, and also indicate the necessity of using multiplex diagnostic tools to increase the sensitivity and specificity for diagnostic tests. Emerging data from the T. cruzi genome and from its ORFeome project will also allow the identification of new antigens for this disease detection application.

Comparative study of amplification systems in immunoenzyme assays for the diagnosis of American tegumentary leishmaniasis.

We compared the accuracy and reliability of three amplification systems for enzyme immunoassays in the detection of specific IgG antibodies for the diagnosis of cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in patients from an endemic area in Rio de Janeiro, Brazil. Partially soluble antigens obtained from the promastigote forms of L. (V.) braziliensis were used. For development of the reaction, two chromogens, 1,2-orthophenylenediamine (OPD) and 3,3',5,5'-tetramethylbenzidine (TMB), and a fluorogen, 4-methylumbelliferylphosphate (MUP), were tested. The performance of each system was compared using the following parameters: accuracy, intraclass correlation coefficient (ICC), and area under the receiver operating characteristic (ROC) curve. Sensitivity was the same (97.4%) for all systems. The reliability was excellent (ICC = 98.6, 98.7, and 99.1%) and specificity was 93.7, 95.8, and 97.4% for OPD, MUP, and TMB, respectively, showing no statistical significance. Despite the absence of differences in the performance of the three systems, the use of TMB is suggested because of its operational advantages, such as low cost compared with fluorogens, easy manipulation, greater stability, and lower toxicity.

[Serological evaluation for detection of anti-Leishmania antibodies in dogs and cats in the district of Santa Rita de Cássia, municipality of Barra Mansa, State of Rio de Janeiro]. / Avaliação sorológica para detecção de anticorpos anti-Leishmania em cães e gatos no bairro de Santa Rita de Cássia, Município de Barra Mansa, Estado do Rio de Janeiro.

From a canine case of american cutaneous leishmaniasis in the locality of Santa Rita de Cássia, municipality of Barra Mansa, Rio de Janeiro, a serological survey was performed on 177 dogs and 43 cats. Evaluation of the canine serum samples showed that 10% had a positive reaction in the indirect immunofluorescence test and 10.7% in the enzyme-linked immunosorbent assay. Among the feline serum samples tested, none of the animals had a positive reaction in the indirect immunofluorescence test and only one (2.4%) showed a positive reaction in the enzyme-linked immunosorbent assay. The detection of an autochthonous case of Leishmania braziliensis in Barra Mansa gives warning that a focus of american cutaneous leishmaniasis is possibly becoming established in this region.

Avaliação sorológica para detecção de anticorpos anti-Leishmania em cães e gatos no bairro de Santa Rita de Cássia, Município de Barra Mansa, Estado do Rio de Janeiro / Serological evaluation for detection of anti-Leishmania antibodies in dogs and cats in the district of Santa Rita de Cássia, municipality of Barra Mansa, State of Rio de Janeiro

A partir de um caso canino de leishmaniose tegumentar americana na localidade de Santa Rita de Cássia, município de Barra Mansa, Rio de Janeiro, foi realizado um inquérito sorológico em 177 cães e 43 gatos. Na avaliação das amostras de soros caninos, 10 por cento foram positivos na reação de imunofluorescência indireta e 10,7 por cento no ensaio imunoenzimático. Entre as amostras de soros felinos testados, nenhum animal foi positivo na reação de imunofluorescência indireta e apenas um (2,4 por cento) felino apresentou reação positiva ao ensaio imunoenzimático. A detecção de Leishmania braziliensis, autóctone em Barra Mansa, faz um alerta para a instalação de um possível foco de leishmaniose tegumentar americana nessa região.

From a canine case of american cutaneous leishmaniasis in the locality of Santa Rita de Cássia, municipality of Barra Mansa, Rio de Janeiro, a serological survey was performed on 177 dogs and 43 cats. Evaluation of the canine serum samples showed that 10 percent had a positive reaction in the indirect immunofluorescence test and 10.7 percent in the enzyme-linked immunosorbent assay. Among the feline serum samples tested, none of the animals had a positive reaction in the indirect immunofluorescence test and only one (2.4 percent) showed a positive reaction in the enzyme-linked immunosorbent assay. The detection of an autochthonous case of Leishmania braziliensis in Barra Mansa gives warning that a focus of american cutaneous leishmaniasis is possibly becoming established in this region.

Differentiation between canine cutaneous and visceral leishmaniasis by the detection of immunoglobulin G specific for Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi antigens using flow cytometry.

Flow cytometry employing Leishmania (L.) chagasi (Lc) and L. (Viannia) braziliensis (Lb) antigen was used to establish the differential diagnosis between visceral (VL) and cutaneous leishmaniasis (CL) in dogs. Flow cytometry permitted the detection of Leishmania-specific immunoglobulin G in sera from 19 dogs: nine with CL and 10 with VL. A significant difference in the percentage of positive staining was observed in sera from dogs with CL between the homologous antigen (69% for Lb) and the heterologous antigen (42% for Lc). However, this difference was not significant in sera from dogs with VL (61% for Lb and 73% for Lc). No significant staining was observed in control sera (0.6% for Lb and 0.4% for Lc) consisting of samples from healthy dogs, or in the group with sporotrichosis (1.8% for Lb and 1.5% for Lc), a differential diagnosis of CL. The results suggest that flow cytometry might be useful for the differentiation between CL and VL in dogs, with practical applications in areas where the two infections overlap.