Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples

Losada-Barragán, Mónica; Cavalcanti, Amanda dos Santos; Umaña‐Pérez, Adriana; Porrozzi, Renato; Cuervo-Escobar, Sergio; Vallejo, Andrés F.; Sánchez-Gómez, Myriam; Cuervo, Patrícia

doi:10.1016/j.vetpar.2016.05.022

Cited by 9 publications

(9 citation statements)

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“…Parasites were detected in the liver and the spleen by a qPCR assay with a detection limit of 0.1 parasites per 10 6 cells, as described before24. qPCR determined that 100% liver and spleen samples were positive for the infection (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Positivity of infection and parasite load in the liver and spleen were measured by real-time quantitative PCR (qPCR) following procedures previously described2224. In brief, total DNA was extracted from the liver and spleen using the Wizard Genomic DNA Purification System (Promega, Madison, WI, USA), which includes a prior digestion phase with 17.5 μl of proteinase K (20 mg/mL) for 12 h at 55 °C.…”

Section: Methodsmentioning

confidence: 99%

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Protein malnutrition promotes dysregulation of molecules involved in T cell migration in the thymus of mice infected with Leishmania infantum

Losada-Barragán

Umaña‐Pérez

Cuervo-Escobar

et al. 2017

Sci Rep

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Protein malnutrition, the most deleterious cause of malnutrition in developing countries, has been considered a primary risk factor for the development of clinical visceral leishmaniasis (VL). Protein malnutrition and infection with Leishmania infantum leads to lymphoid tissue disorganization, including changes in cellularity and lymphocyte subpopulations in the thymus and spleen. Here we report that protein malnutrition modifies thymic chemotactic factors by diminishing the CCL5, CXCL12, IGF1, CXCL9 and CXCL10 protein levels in infected animals. Nevertheless, T cells preserve their migratory capability, as they were able to migrate ex vivo in response to chemotactic stimuli, indicating that malnutrition may compromise the thymic microenvironment and alter in vivo thymocyte migration. Decrease in chemotactic factors protein levels was accompanied by an early increase in the parasite load of the spleen. These results suggest that the precondition of malnutrition is affecting the cell-mediated immune response to L. infantum by altering T cell migration and interfering with the capacity of protein-deprived animals to control parasite spreading and proliferation. Our data provide evidence for a disturbance of T lymphocyte migration involving both central and peripheral T-cells, which likely contribute to the pathophysiology of VL that occurs in malnourished individuals.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Protein malnutrition promotes dysregulation of molecules involved in T cell migration in the thymus of mice infected with Leishmania infantum

Losada-Barragán

Umaña‐Pérez

Cuervo-Escobar

et al. 2017

Sci Rep

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“…Since HRM analysis resulted inconclusive for discrimination of L. ( L. ) infantum and L. ( L. ) amazonensis in about 53% of samples, a new SYBR-green qPCR assay (qPCR-ama) was designed to amplify a minicircle subclass preponderant in L. ( L. ) amazonensis, rather than targeting a hypothetical species-specific sequence. In fact, several PCR assays designed on minicircles also reported amplification of non-intended species [17, 29]. Moreover, Gomes et al [30] showed that non-target organisms such as T. cruzi could be amplified, even if C q values are ˃ 30.…”

Section: Discussionmentioning

confidence: 99%

“…However, the minicircle network is composed of different minicircle subclasses conserved in different Leishmania species [15, 16]. Therefore, most of the available PCR or qPCR assays designed for a single species can potentially amplify more than one Leishmania species [13, 14, 17]. …”

Section: Introductionmentioning

confidence: 99%

The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis

et al. 2017

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BackgroundLeishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area.MethodsDNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed.ResultsThe qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the Cq values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of Cq values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples.ConclusionsA new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a minicircle sequence rather than targeting a hypothetical species-specific sequence. The fast and accurate discrimination between these species can be useful to provide adequate prognosis and treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2181-x) contains supplementary material, which is available to authorized users.

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“…PCR primers that specifically amplify a species or a group of species have been designed on minicircle kDNA, exploiting sequence polymorphisms. However, several authors observed cross-reaction among species, probably because of minicircle classes variability in each of them [ 17 , 19 , 20 , 23 ]. In a different approach, using common primers to amplify different species, differences in melting temperature (Tm) of PCR-amplified minicircle kDNA regions were exploited to differentiate Leishmania species.…”

Section: Qpcr Assays For Leishmania Genotypingmentioning

confidence: 99%

Real-time PCR applications for diagnosis of leishmaniasis

et al. 2018

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Leishmaniasis is a vector-borne disease caused by many Leishmania species, which can infect both humans and other mammals. Leishmaniasis is a complex disease, with heterogeneous clinical manifestations ranging from asymptomatic infections to lesions at cutaneous sites (cutaneous leishmaniasis), mucosal sites (mucocutaneous leishmaniasis) or in visceral organs (visceral leishmaniasis), depending on the species and host characteristics. Often, symptoms are inconclusive and leishmaniasis can be confused with other co-endemic diseases. Moreover, co-infections (mainly with HIV in humans) can produce atypical clinical presentations. A correct diagnosis is crucial to apply the appropriate treatment and the use of molecular techniques in diagnosis of leishmaniasis has become increasingly relevant due to their remarkable sensitivity, specificity and possible application to a variety of clinical samples. Among them, real-time PCR (qPCR)-based approaches have become increasingly popular in the last years not only for detection and quantification of Leishmania species but also for species identification. However, despite qPCR-based methods having proven to be very effective in the diagnosis of leishmaniasis, a standardized method does not exist. This review summarizes the qPCR-based methods in the diagnosis of leishmaniasis focusing on the recent developments and applications in this field.

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Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples

Cited by 9 publications

References 50 publications

Protein malnutrition promotes dysregulation of molecules involved in T cell migration in the thymus of mice infected with Leishmania infantum

Protein malnutrition promotes dysregulation of molecules involved in T cell migration in the thymus of mice infected with Leishmania infantum

The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis

Real-time PCR applications for diagnosis of leishmaniasis

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