Quantification of Leishmania Infantum Parasites in Tissue Biopsies by Real-Time Polymerase Chain Reaction and Polymerase Chain Reaction–enzyme-Linked Immunosorbent Assay

Rolão, Nuno; Cortes, Sofia; Rodrigues, Olı́via Roos; Campino, Lenea

doi:10.1645/ge-264r1

Cited by 64 publications

(39 citation statements)

References 27 publications

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“…Parasite density was determined using realtime PCR following the protocol described by EVALUATION OF TNF-a, IL-4, AND IL-10 AND PARASITE DENSITY IN INFECTED DOGS Rolão et al (2004). To achieve this, 35 mg of spleen or liver from each dog were used for DNA extraction with a commercial kit (PCR template Preparation Kit, Roche Diagnostics GmbH, Mannheim, Germany), in accordance with the manufacturer's instructions.…”

Section: Parasite Loadmentioning

confidence: 99%

“…These dilutions were then used for DNA extraction, as described above. Primers and internal probes were designed according to regions of the kinetoplast minicircle of L. (L.) chagasi (GenBank accession number AF169140), in accordance with Rolão et al (2004). DNA samples were analyzed with the following (Applied Byosystems, Foster City, CA, USA): direct primer LshNRf (forward, 59-GGTTAGCCGATGGTGG-TCTT-39), reverse LshNRr (59-GCTAT-ATCATATGTCCAAGCACTTACCT-39), and internal probe LshNRp TaqManH (59-ACCACCTAAGGTCAACCC-39).…”

Section: Parasite Loadmentioning

confidence: 99%

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Evaluation of TNF-α, IL-4, and IL-10 and parasite density in spleen and liver ofL. (L.) chagasinaturally infected dogs

Michelin

Perri

Lima

2011

Annals of Tropical Medicine & Parasitology

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Dogs are the main domestic reservoirs of L. (L.) chagasi. Once in the vertebrate host, the parasite can cause visceral leishmaniasis, which can also be transmitted to humans. Cytokines are key elements of the host immune response against Leishmania spp. To investigate whether tumor necrosis factor (TNF)-a, interleukin (IL)-4 and IL-10 are associated with pattern infection in dogs, these cytokines were quantified in the spleen and liver of dogs naturally infected with L. (L.) chagasi, with or without clinical manifestations, and their levels were correlated with the parasite load verified in these organs. A total of 40 adult dogs naturally infected with L. (L.) chagasi were assessed, together with 12 uninfected control dogs. Samples from spleen and liver were used to determine the cytokine levels by capture ELISA and for quantifying parasite load by real-time PCR. Statistical analysis was performed using the minimum Chi square method and group means were compared using the Tukey test. TNF-a, IL-4 and IL-10 levels in infected dogs were higher than in control groups; the liver was the main cytokine-producing organ during infection. The level of splenic TNF-a showed correlation with parasite load and may represent an important marker for infection process evolution, with the participation of IL-10. These results may contribute to a clearer understanding of the immune response in dogs infected with L. (L.) chagasi, which may lead to the development of prophylactic or preventive measures for these animals.

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Section: Parasite Loadmentioning

confidence: 99%

Section: Parasite Loadmentioning

confidence: 99%

Evaluation of TNF-α, IL-4, and IL-10 and parasite density in spleen and liver ofL. (L.) chagasinaturally infected dogs

Michelin

Perri

Lima

2011

Annals of Tropical Medicine & Parasitology

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“…Several diagnostic tools have been applied to detect this infection in dogs, including parasitological, serological (MOREIRA et al, 2007) and molecular techniques (ROLÃO et al, 2004). In Brazil, the official diagnosis is based on serological tests, i.e.…”

Section: Introductionmentioning

confidence: 99%

Quantification of Leishmania infantumDNA in the bone marrow, lymph node and spleen of dogs

Ramos¹,

Ramos

Santos

et al. 2013

Rev. Bras. Parasitol. Vet.

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The aim of the present study was to quantify the parasite load of Leishmania infantum in dogs using real-time PCR (qPCR). Bone marrow, lymph node and spleen samples were taken from 24 dogs serologically positive for L. infantum that had been put down by the official epidemiological surveillance service. According to the clinical signs the dogs were classified as asymptomatic or symptomatic. After DNA extraction, the samples were subjected to qPCR to detect and quantify L. infantum DNA. Out of the 24 dogs, 12.5% (3/24) were classified as asymptomatic and 87.5% (21/24) as symptomatic. Real-time PCR detected L. infantum DNA in all the animals, in at least one biological sample. In particular, 100% of bone marrow and lymph node scored positive, whereas in spleen, the presence of DNA was detected in 95.9% (23/24). In addition, out of 24 animals, 15 were microscopically positive to amastigote forms of L. infantum in bone marrow. No statistical significant difference was found in the overall mean quantity of DNA among the different biological samples (P = 0.518). Considering each organ separately, there was 100% positivity in bone marrow and lymph nodes, while among the spleen samples, 95.9% (23/24) were positive. Regarding the different clinical groups, the overall mean parasite load varied significantly (P = 0.022). According to the results obtained, it was not possible determine which biological sample was most suitable tissue for the diagnosis, based only on the parasite load. Therefore, other characteristics such as convenience and easily of obtaining samples should be taken into consideration.Keywords: Leishmania, molecular, diagnosis, parasite load, dogs. ResumoO objetivo do presente estudo foi quantificar a carga parasitária de Leishmania infantum em cães pela técnica de PCR em tempo-real (qPCR). Amostras de medula óssea, linfonodo e baço foram obtidos de 24 cães sorologicamente positivos para L. infantum que foram submetidos à eutanásia pelo serviço de vigilância oficial. Segundo os sinais clínicos, os animais foram classificados em assintomáticos ou sintomáticos. Após extração de DNA, as amostras foram submetidas à qPCR para detecção e quantificação de DNA de L. infantum. Dos 24 cães, 12,5% (3/24) foram classificados como assintomáticos e 87,5% (21/24) como sintomáticos. A PCR em tempo real detectou DNA de L. infantum em 100% dos animais, em pelo menos uma amostra biológica. Considerando cada órgão isoladamente, foi observada uma positividade de 100% em medula óssea e linfonodo, já nas amostras de baço 95,9% (23/24) foram positivas. Não foi observada diferença estatística entre a quantidade média geral de DNA entre as diferentes amostras biológicas (P = 0,518). Considerando os diferentes grupos clínicos, a carga parasitária média geral variou significantemente (P = 0,022). De acordo com os resultados obtidos não foi possível eleger a mais apropriada amostra biológica para o diagnóstico, baseado apenas na carga parasitária. Portanto, outras características como a conveniência e a facilidade de obtenção...

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“…Moreover, restriction enzyme analysis after amplification allows differentiation between different Leishmania species (2,10,25). Currently, different DNA and RNA amplification methods have been established for detection and quantification of Leishmania parasites, including quantitative real-time PCR (qPCR) and quantitative nucleic acid sequence-based amplification (QT-NASBA) (8,21,27,32). Sensitive molecular tools can offer significant advantages, not only in diagnosis but also in studies requiring accurate and sensitive quantification of parasites, such as drug treatment efficacy studies (7).…”

mentioning

confidence: 99%

Comparison between Quantitative Nucleic Acid Sequence-Based Amplification, Real-Time Reverse Transcriptase PCR, and Real-Time PCR for Quantification ofLeishmaniaParasites

et al. 2008

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DNA or RNA amplification methods for detection of Leishmania parasites have advantages regarding sensitivity and potential quantitative characteristics in comparison with conventional diagnostic methods but are often still not routinely applied. However, the use and application of molecular assays are increasing, but comparative studies on the performance of these different assays are lacking. The aim of this study was to compare three molecular assays for detection and quantification of Leishmania parasites in serial dilutions of parasites and in skin biopsies collected from cutaneous leishmaniasis (CL) patients in Manaus, Brazil. A serial dilution of promastigotes spiked in blood was tested in triplicate in three different runs by quantitative nucleic acid sequence-based amplification (QT-NASBA), quantitative real-time reverse transcriptase PCR (qRT-PCR), and quantitative real-time PCR (qPCR). In addition, the costs, durations, and numbers of handling steps were compared, and 84 skin biopsies from patients with suspected CL were tested. Both QT-NASBA and qRT-PCR had a detection limit of 100 parasites/ml of blood, while qPCR detected 1,000 parasites/ml. QT-NASBA had the lowest range of intra-assay variation (coefficients of variation [CV], 0.5% to 3.3%), while qPCR had the lowest range of interassay variation (CV, 0.4% to 5.3%). Furthermore, qRT-PCR had higher r 2 values and amplification efficiencies than qPCR, and qPCR and qRT-PCR had faster procedures than QT-NASBA. All assays performed equally well with patient samples, with significant correlations between parasite counts. Overall, qRT-PCR is preferred over QT-NASBA and qPCR as the most optimal diagnostic assay for quantification of Leishmania parasites, since it was highly sensitive and reproducible and the procedure was relatively fast.

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Quantification of Leishmania Infantum Parasites in Tissue Biopsies by Real-Time Polymerase Chain Reaction and Polymerase Chain Reaction–enzyme-Linked Immunosorbent Assay

Cited by 64 publications

References 27 publications

Evaluation of TNF-α, IL-4, and IL-10 and parasite density in spleen and liver ofL. (L.) chagasinaturally infected dogs

Evaluation of TNF-α, IL-4, and IL-10 and parasite density in spleen and liver ofL. (L.) chagasinaturally infected dogs

Quantification of Leishmania infantumDNA in the bone marrow, lymph node and spleen of dogs

Comparison between Quantitative Nucleic Acid Sequence-Based Amplification, Real-Time Reverse Transcriptase PCR, and Real-Time PCR for Quantification ofLeishmaniaParasites

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