Canine visceral leishmaniasis: study of methods for the detection of IgG in serum and eluate samples

Figueiredo, Fabiano Borges; Madeira, M.F.; Nascimento, Lílian Dias; Abrantes, Tuanne Rotti; Mouta-Confort, Eliame; Passos, Sônia Regina Lambert; Schubach, Tânia Maria Pacheco

doi:10.1590/s0036-46652010000400005

Cited by 25 publications

(22 citation statements)

References 21 publications

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“…The performance observed for the synthetic antigens in this work is consistent with the performance of many antigens (mainly recombinant proteins) that have been developed in recent years for CVL diagnosis by ELISA (23,24), with the advantages of being cheaper, simpler, reproducible, useful for large-scale testing and, in most cases, more specific and sensitive (26). Our results show an improvement in the sensitivity of the synthetic antigen ELISA, compared to the indirect immunofluorescence assay, in asymptomatic dogs, due to the fact that the IFAT shows a lack of sensitivity for the detection of specific antibodies in asymptomatic dogs (20) and in healthy dogs, due to the large number of false-positive IFAT reactions (18). It should be taken into account that the ELISA technique allows better interpretation of the results than IFAT, because in that technique the interpretation is subjective and depends on the expertise of the operator.…”

Section: Discussionmentioning

confidence: 74%

“…In this context, in areas in which CVL is endemic, about 10 to 62% of apparently healthy and/or seronegative dogs are positive for Leishmania by PCR (14)(15)(16)(17). Thus, asymptomatic dogs, which play a role in the transmission of parasites, are not detected by conventional serological tests, such as the indirect fluorescent antibody test (IFAT) and the enzymelinked immunosorbent assay (ELISA) (18). Nonetheless, the detection of asymptomatic CVL might be crucial in controlling epidemics and avoiding the spread of disease among dogs, as well as between dogs and human populations (19,20).…”

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confidence: 99%

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Sensitive and Specific Serodiagnosis of Leishmania infantum Infection in Dogs by Using Peptides Selected from Hypothetical Proteins Identified by an Immunoproteomic Approach

Chávez‐Fumagalli

Martins

Testasicca

et al. 2013

Clin Vaccine Immunol

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In Brazil, the percentage of infected dogs living in areas where canine visceral leishmaniasis (CVL) is endemic ranges from 10 to 62%; however, the prevalence of infection in dogs is probably higher than figures reported from serological studies. In addition, problems with the occurrence of false-positive or false-negative results in the serodiagnosis of CVL have been reported. The present work analyzed the potential of synthetic peptides mapped from hypothetical proteins for improvement of the serodiagnosis of Leishmania infantum infection in dogs. From 26 identified leishmanial proteins, eight were selected, considering that no homologies between these proteins and others from trypanosomatide sequence databases were encountered. The sequences of these proteins were mapped to identify linear B-cell epitopes, and 17 peptides were synthesized and tested in enzyme-linked immunosorbent assays (ELISAs) for the serodiagnosis of L. infantum infection in dogs. Of these, three exhibited sensitivity and specificity values higher than 75% and 90%, respectively, to differentiate L. infantum-infected animals from Trypanosoma cruziinfected animals and healthy animals. Soluble Leishmania antigen (SLA) showed poor sensitivity (4%) and specificity (36%) to differentiate L. infantum-infected dogs from healthy and T. cruzi-infected dogs. Lastly, the three selected peptides were combined in different mixtures and higher sensitivity and specificity values were obtained, even when sera from T. cruzi-infected dogs were used. The study's findings suggest that these three peptides can constitute a potential tool for more sensitive and specific serodiagnosis of L. infantum infection in dogs.

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Section: Discussionmentioning

confidence: 74%

mentioning

confidence: 99%

Sensitive and Specific Serodiagnosis of Leishmania infantum Infection in Dogs by Using Peptides Selected from Hypothetical Proteins Identified by an Immunoproteomic Approach

Chávez‐Fumagalli

Martins

Testasicca

et al. 2013

Clin Vaccine Immunol

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“…Em áreas endêmicas, o Ministério da Saúde recomenda a eutanásia dos cães reativos na RIFI a partir da diluição de 1:40 (Figueiredo et al 2010). Esses autores observaram que a sensibilidade da RIFI foi igual para os dois pontos de corte aplicados (1:40 e 1:80).…”

Section: Discussionunclassified

Diagnóstico molecular e frequência de anticorpos anti-Leishmania infantum chagasi em cães do município de Belém, Pará

et al. 2014

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A leishmaniose visceral é uma enfermidade cujo agente etiológico no Brasil é o protozoário Leishmania infantum chagasi. Os cães são considerados reservatórios urbanos da doença, sendo indicadores da ocorrência de casos humanos. O presente trabalho teve como objetivo diagnosticar a infecção por L. infantum chagasi em cães domiciliados e errantes do município de Belém, estado do Pará, através da reação em cadeia da polimerase (PCR) e da reação de imunofluorescência indireta (RIFI), empregando dois antígenos distintos. Amostras de sangue venoso de cães adultos, sem distinção de sexo ou raça, de diferentes bairros e épocas do ano da cidade de Belém-PA, foram colhidas em tubos sem e com anticoagulante para obtenção do soro e do DNA, respectivamente. Esses animais foram divididos em dois grupos: cães errantes capturados pelo Centro de Controle de Zoonoses (Grupo A) e cães domiciliados (Grupo B). Os soros foram analisados através do teste de RIFI para pesquisa de IgG utilizando-se dois antígenos distintos: 1) antígeno do kit Bio-Manguinhos/FIOCRUZ (Ag-PRO) contendo formas promastigotas de Leishmania sp. (complexo major-like); 2) Antígeno do Instituto Evandro Chagas (Ag-AMA) constituído por formas amastigotas de L. infantum chagasi. A avaliação dos dois antígenos foi realizada com as amostras reagentes a partir da titulação 1:80. Já a PCR foi realizada a partir do DNA extraído do sangue total dos animais e amplificado utilizando-se os iniciadores RV1e RV2. Das 335 amostras analisadas, 10,4% (35/335) foram reagentes na RIFI (Ag-PRO) e 0,9% (3/335) reagiram com o Ag-AMA. A distribuição das amostras positivas se deu da seguinte forma: Grupo A 14,8% (25/169) com Ag-PRO e 1,2% (2/169) com Ag-AMA; Grupo B 6% (10/166) com Ag-PRO e 0,6% (1/166) com Ag-AMA; sendo que todas as amostras positivas pelo teste de RIFI com o Ag-AMA também reagiram com o Ag-PRO e em nenhuma das amostras foi detectado o DNA de L. infantum chagasi. Os achados do presente estudo indicam que Belém ainda pode ser considerada área não endêmica para leishmaniose visceral canina e que a natureza do antígeno influencia no resultado da RIFI para a pesquisa de anticorpos anti-L. infantum chagasi em cães, sendo que a RIFI que utiliza formas promastigotas de Leishmania major-like como antígeno deve ser utilizada com cautela como método diagnóstico confirmatório em estudos epidemiológicos em áreas não endêmicas para LVC.

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“…In addition, crude antigens are also limited by the difficulty in producing large quantities in a standardized manner. Moreover, a high percentage of dogs, though infected by Leishmania, can remain seronegative for various periods of time, which can present as falsenegative results in the serological trials (7,51,52).…”

Section: Discussionmentioning

confidence: 99%

“…CVL is also an important concern in countries that are not endemic for the disease, as imported sick or infected dogs constitute a veterinary and public health problem (6). Brazilian public health authorities currently use the enzyme-linked immunosorbent assay (ELISA) as a screening test and the indirect immunofluorescence antibody test (IFAT), with a cutoff titer of 1:40, as a confirmatory analysis to determine the clinical status of dogs (7). Moreover, controlling the spread of CVL in Brazil is mainly based on the elimination of seropositive dogs (8).…”

mentioning

confidence: 99%

Subtractive Phage Display Selection from Canine Visceral Leishmaniasis Identifies Novel Epitopes That Mimic Leishmania infantum Antigens with Potential Serodiagnosis Applications

Costa¹,

Lima²,

Chávez-Fumagalli³

et al. 2013

Clin. Vaccine Immunol.

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i Visceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy and Trypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected with Leishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n ‫؍‬ 31) compared to those from vaccinated dogs (n ‫؍‬ 21), experimentally infected dogs with cross-reactive parasites (n ‫؍‬ 23), and healthy controls (n ‫؍‬ 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no crossreactivity with T. cruzi-or Ehrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes of L. infantum antigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programs.

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Canine visceral leishmaniasis: study of methods for the detection of IgG in serum and eluate samples

Cited by 25 publications

References 21 publications

Sensitive and Specific Serodiagnosis of Leishmania infantum Infection in Dogs by Using Peptides Selected from Hypothetical Proteins Identified by an Immunoproteomic Approach

Sensitive and Specific Serodiagnosis of Leishmania infantum Infection in Dogs by Using Peptides Selected from Hypothetical Proteins Identified by an Immunoproteomic Approach

Diagnóstico molecular e frequência de anticorpos anti-Leishmania infantum chagasi em cães do município de Belém, Pará

Subtractive Phage Display Selection from Canine Visceral Leishmaniasis Identifies Novel Epitopes That Mimic Leishmania infantum Antigens with Potential Serodiagnosis Applications

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