Molecular diagnosis of canine visceral leishmaniasis: Identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR

Quaresma, Patrícia Flávia; Murta, Silvane Maria Fonseca; Ferreira, Eduardo; Rocha-Lima, Ana Cristina Vianna Mariano da; Xavier, Ana Amélia Prates; Gontijo, Célia Maria Ferreira

doi:10.1016/j.actatropica.2009.05.008

Cited by 89 publications

(63 citation statements)

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“…The tendency towards higher co-positivity percentages in symptomatic dogs compared with asymptomatic dogs, which was consistently observed in this study, agrees with the literature for several diagnostic tests 2,3,[34][35][36][37][38][39] . Depending on the assay, this may be related to higher antibody titers or to higher parasite loads in clinically ill dogs 2 .…”

Section: Discussionsupporting

confidence: 81%

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Evaluation of parasitological examination, kDNA polymerase chain reaction and rK39-based immunochromatography for the diagnosis of visceral leishmaniasis in seropositive dogs from the screening-culling program in Brazil

Regina-Silva

Fortes-Dias

Michalsky

et al. 2014

Rev. Soc. Bras. Med. Trop.

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Introduction: Dogs play a primary role in the zoonotic cycle of visceral leishmaniasis (VL). Therefore, the accurate diagnosis of infected dogs, primarily asymptomatic dogs, is crucial to the effi ciency of VL control programs. Methods: We investigated the agreement of four diagnostic tests for canine visceral leishmaniasis (CVL): parasite detection, either after myeloculture or by direct microscopic examination of tissue imprints; kinetoplast-deoxyribonucleic acid-polymerase chain reaction (kDNA-PCR); and an immunochromatographic test (ICT). An enzyme-linked immunosorbent assay (ELISA) and an indirect immunofl uorescence test (IFAT), both of which were adopted as part of the screening-culling program in Brazil, were used as reference tests. Our sample set consisted of 44 seropositive dogs, 25 of which were clinically asymptomatic and 19 were symptomatic for CVL according to ELISA-IFAT. Results: The highest and lowest test co-positivities were observed for ICT (77.3%) and myeloculture (58.1%), respectively. When analyzed together, the overall percentage of co-positive tests was signifi cantly higher for the symptomatic group compared to the asymptomatic group. However, only ICT was signifi cantly different based on the results of a separate analysis per test for each group of dogs. The majority (93.8%) of animals exhibited at least one positive test result, with an average of 2.66 positive tests per dog. Half of the symptomatic dogs tested positive for all four tests administered. Conclusions:The variability between test results reinforces the need for more effi cient and reliable methods to accurately diagnose canine VL, particularly in asymptomatic animals.

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Section: Discussionsupporting

confidence: 81%

“…Infected dogs may present intense cutaneous parasitism 2,3 and play a primary role in the maintenance of vector infection 4 . Along with measures to control the vector population and early treatment of human cases, euthanasia of infected dogs is part of the VL control policy adopted by the Brazilian Ministry of Health 5 .…”

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confidence: 99%

Evaluation of parasitological examination, kDNA polymerase chain reaction and rK39-based immunochromatography for the diagnosis of visceral leishmaniasis in seropositive dogs from the screening-culling program in Brazil

Regina-Silva

Fortes-Dias

Michalsky

et al. 2014

Rev. Soc. Bras. Med. Trop.

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“…Species of Leishmania other than L. infantum, such as L. amazonensis and L. braziliensis, have previously been isolated from dogs (4,13,22,25,31), although the epidemiological significance of the dog as a reservoir host for these parasites is unclear. Mixed infections with L. braziliensis and L. infantum have also been documented (14).…”

Section: Discussionmentioning

confidence: 99%

Comparison of Leishmania OligoC-TesT PCR with Conventional and Real-Time PCR for Diagnosis of Canine Leishmania Infection

et al. 2010

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There is a need for standardization and simplification of the existing methods for molecular detection of Leishmania infantum in the canine reservoir host. The commercially available OligoC-TesT kit incorporates standardized PCR reagents with rapid oligochromatographic dipstick detection of PCR products and is highly sensitive for use in humans but not yet independently validated for use in dogs. Here we compare the sensitivity of OligoC-TesT with those of nested kinetoplast DNA (kDNA) PCR, nested internal transcribed spacer 1 (ITS-1) PCR, and a PCR-hybridization protocol, using longitudinal naturally infected canine bone marrow samples whose parasite burdens were measured by real-time quantitative PCR (qPCR). The sensitivity of OligoC-TesT for infected dogs was 70% (95% confidence interval [CI], 63 to 78%), similar to that of kDNA PCR (72%; 95% CI, 65 to 80%; P ‫؍‬ 0.69) but significantly greater than those of PCR-hybridization (61%; 95% CI, 53 to 69%; P ‫؍‬ 0.007) and ITS-1 nested PCR (54%; 95% CI, 45 to 62%; P < 0.001); real-time qPCR had the highest sensitivity (91%; 95% CI, 85 to 95%; P < 0.001). OligoC-TesT sensitivity was greater for polysymptomatic and oligosymptomatic dogs than for asymptomatic dogs (93%, 74%, and 61%, respectively; P ‫؍‬ 0.005), a trend also observed for the other qualitative PCR methods tested (P < 0.05). Test positivity increased with increasing parasite burdens, as measured by real-time qPCR: OligoC-TesT and kDNA PCR detected 100% and 99% of positive samples when parasite burdens exceeded 74 and 49 parasites/ml, respectively. OligoC-TesT has high sensitivity for detection of canine Leishmania infections; its ease of operation and ease of interpretation are further advantages for veterinary diagnostic laboratories and for large-scale survey work in developing countries.

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“…A padronização da PCR realizada a partir de amostras de sangue total de dois cães sintomá-ticos, positivos no exame parasitológico e reagentes na sorologia, detectou DNA de L. infantum chagasi até a diluição do DNA de 1:10, demostrando uma ótima capacidade diagnóstica em amostras de sangue total de cães sintomáticos, corroborando com os dados obtidos por outros autores (Lachaud et al 2002, Gomes et al 2007, Quaresma et al 2009, Leite et al 2010.…”

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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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Molecular diagnosis of canine visceral leishmaniasis: Identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR

Cited by 89 publications

References 35 publications

Evaluation of parasitological examination, kDNA polymerase chain reaction and rK39-based immunochromatography for the diagnosis of visceral leishmaniasis in seropositive dogs from the screening-culling program in Brazil

Evaluation of parasitological examination, kDNA polymerase chain reaction and rK39-based immunochromatography for the diagnosis of visceral leishmaniasis in seropositive dogs from the screening-culling program in Brazil

Comparison of Leishmania OligoC-TesT PCR with Conventional and Real-Time PCR for Diagnosis of Canine Leishmania Infection

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

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