Performance of a real time PCR for leishmaniasis diagnosis using a L. (L.) infantum hypothetical protein as target in canine samples

Colombo, Fábio Antônio; Pereira-Chioccola, Vera Lúcia; Meira, Cristina da Silva; Motoie, Gabriela; Gava, Ricardo; Hiramoto, Roberto Mitsuyoshi; Almeida, Marcos E. de; Silva, Alexandre J. da; Cutolo, André Antonio; Menz, Ingrid

doi:10.1016/j.exppara.2015.08.014

Cited by 19 publications

(16 citation statements)

References 40 publications

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“…For sensitive and accurate quantification of parasites in infected tissues, we used a quantitative reverse transcription PCR (qRT-PCR) assay based on the detection of the LINJ31 marker (Linj31-qPCR), which was used successfully for the quantification of live amastigotes of Leishmania (L.) infantum chagasi [4,9,15,16]. The use of RNA as a sample, rather than DNA, minimizes the detection of residual DNA from dead parasites [8,16]. Similar qPCR-based tests allow effective quantification of parasite genetic material from the spleen and liver of infected hamsters [4,9] and yield reliable estimates of the parasite burden per gram of tissue [9,17].…”

Section: Resultsmentioning

confidence: 99%

“…Standard curves of parasite DNA for use in quantitative real-time PCR (qPCR) experiments were produced as described previously [8]. Briefly, promastigotes from stationary phase cultures were harvested by centrifugation at 1000 g for 10 minutes, washed twice in PBS (pH 7.2), and counted in a hemocytometer.…”

Section: Dna Extractionmentioning

confidence: 99%

“…To improve treatment monitoring, the detection and quantification of amastigote forms was performed by quantitative real-time PCR (qPCR). When compared to conventional PCR, qPCR represents a more efficient, sensitive and robust technique for the detection of amastigotes RNA in various tissues (spleen, skin, lymph, blood and bone marrow) and in different clinical groups [7][8][9]. In addition, qPCR is currently considered the most reliable method to evaluate the in vivo activity of compounds against visceral leishmaniasis, allowing effective disease monitoring, with increased speed and accuracy compared with standard methods [10,11].…”

Section: Introductionmentioning

confidence: 99%

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In Vivo Evaluation of Leishmanicidal Activity of Benzophenone Derivatives by qPCR

Colombo¹,

Reis²,

Nunes³

et al. 2017

Med chem

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In many tropical areas, infections by Leishmania species are endemic and include visceral leishmaniasis (VL), which is often fatal if untreated. Outside India, VL treatment and control are based on long-term administration of highly toxic pentavalent antimonials. Previously, we described the synthesis and in vitro leishmanicidal activity of a series of nine benzophenone derivatives with low toxicity towards murine macrophages. Here in, we report the in vivo evaluation of the most promising active compounds of that series in an experimental model of established VL by L. (L). infantum chagasi in hamsters. Importantly, parasite DNA (amastigote form) quantification in infected tissues was performed by real time PCR, for improved detection accuracy and speed. Compounds 2-Hydroxy-4-O-(3,3-dimethyl)-allylbenzophenone (LFQM-117 (1)), 4-O-(3,3-Dimethyl)-allylbenzophenone (LFQM-120 (2)) and 4,4′-Di-methoxybenzophenone (LFQM-121 (3)) were administered as oral suspensions (50 mg/kg/day)

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Section: Resultsmentioning

confidence: 99%

Section: Dna Extractionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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In Vivo Evaluation of Leishmanicidal Activity of Benzophenone Derivatives by qPCR

Colombo¹,

Reis²,

Nunes³

et al. 2017

Med chem

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“…simultaneously, many of the described qPCR methods detect only some of the pertinent Leishmania spp. or strains (24,27,28,30). Although assays that target minicircle kinetoplast DNA (kDNA) are the most sensitive PCR methods for detecting Leishmania parasites because of the abundance of minicircles in each kinetoplast (13,29,31,32), the high-level sequence polymorphism among minicircles is a substantial limitation for systematic species identification solely on the basis of this marker.…”

mentioning

confidence: 99%

“…The advantages of real-time quantitative PCR (qPCR) approaches in comparison with conventional PCR assays include the potential for increased sensitivity and specificity and for a decreased risk of contamination of the laboratory environment (14,(18)(19)(20)(21)(22)(23)(24)(25)(26). The efficiency of real-time platforms for Leishmania species identification has been discussed (24,(27)(28)(29)(30). Although real-time PCR approaches have the potential for identifying different Leishmania spp.…”

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confidence: 99%

Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay

et al. 2017

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Leishmaniasis in humans is caused by Leishmania spp. in the subgenera Leishmania and Viannia. Species identification often has clinical relevance. Until recently, our laboratory relied on conventional PCR amplification of the internal transcribed spacer 2 (ITS2) region (ITS2-PCR) followed by sequencing analysis of the PCR product to differentiate Leishmania spp. Here we describe a novel real-time quantitative PCR (qPCR) approach based on the SYBR green technology (LSG-qPCR), which uses genus-specific primers that target the ITS1 region and amplify DNA from at least 10 Leishmania spp., followed by analysis of the melting temperature (T m ) of the amplicons on qPCR platforms (the Mx3000P qPCR system [Stratagene-Agilent] and the 7500 real-time PCR system [ABI Life Technologies]). We initially evaluated the assay by testing reference Leishmania isolates and comparing the results with those from the conventional ITS2-PCR approach. Then we compared the results from the real-time and conventional molecular approaches for clinical specimens from 1,051 patients submitted to the reference laboratory of the Centers for Disease Control and Prevention for Leishmania diagnostic testing. Specimens from 477 patients tested positive for Leishmania spp. with the LSG-qPCR assay, specimens from 465 of these 477 patients also tested positive with the conventional ITS2-PCR approach, and specimens from 10 of these 465 patients had positive results because of retesting prompted by LSG-qPCR positivity. On the basis of the T m values of the LSG-qPCR amplicons from reference and clinical specimens, we were able to differentiate four groups of Leishmania parasites: the Viannia subgenus in aggregate; the Leishmania

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Comparative analysis of real-time PCR assays in the detection of canine visceral leishmaniasis

et al. 2018

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Dogs are important hosts and reservoirs of leishmaniasis, a disease caused by protozoan parasites from the genus Leishmania, affecting ~12 million people worldwide. The detection of visceral leishmaniasis (VL) in dogs by real-time PCR (qPCR) may improve on diagnosis, but the different qPCR methods available for Leishmania DNA detection have not been established as routine in diagnostic tools and/or epidemiologic studies for canine VL. Here, we compared three qPCR assays (DNApol, Linj31, and LDON) in the detection of VL by Leishmania infantum in spleen (n = 48; 7), skin (n = 48; 7), and whole blood (n = 44; 7) samples from serologically positive and negative dogs, respectively. Overall, the DNApol performed better than the Linj31 and LDON assays in the detection of positive samples in all tissues tested, yielding from 66.7 to 100.0% of positivity for both skin and spleen samples. For spleen samples, we observed no statistically significant differences between positive detection by the LDON and DNApol assays. Whole blood samples yielded the lowest rates of positive detection, regardless of the qPCR assay used. In contrast, positive detection of Leishmania DNA was as efficient from skin samples using the DNApol assay as from spleen samples using either the DNApol or the LDON assay. Although qPCR assays from skin samples may not be practical for use in the field, our study suggests that the DNApol and LDON assays from skin samples could be used in future to evaluate canine VL treatment in veterinary clinics.

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Performance of a real time PCR for leishmaniasis diagnosis using a L. (L.) infantum hypothetical protein as target in canine samples

Cited by 19 publications

References 40 publications

In Vivo Evaluation of Leishmanicidal Activity of Benzophenone Derivatives by qPCR

In Vivo Evaluation of Leishmanicidal Activity of Benzophenone Derivatives by qPCR

Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay

Comparative analysis of real-time PCR assays in the detection of canine visceral leishmaniasis

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