Application of qPCR in conjunctival swab samples for the evaluation of canine leishmaniasis in borderline cases or disease relapse and correlation with clinical parameters

The Leishmaniases are a group of parasitic diseases caused by protozoa of the Leishmania genus affecting both humans and other vertebrates. Leishmania is an intracellular pathogen able to confer resistance to apoptosis in the early phase of macrophages infection by activation of host PI3K/Akt pathway and inhibition of caspase-3 activation. Intracellular pathogens hijack organelles such as ER to facilitate survival and replication, thus eliciting ER stress and activating/modulating the unfolded protein response (UPR) in the host cell. The UPR is aimed to mitigate ER stress, thereby promoting cell survival. However, prolonged ER stress will activate the apoptotic pathway. The aim of this study was to investigate the ER stress response in Leishmania-infected macrophages to gain insights about the mechanisms underlying the apoptosis resistance in parasitized cells. Macrophages differentiated from human monocytic cell lines (U937 and THP-1) and murine primary macrophages were infected with Leishmania infantum MHOM/TN/80/IPT1 (WHO international reference strain). Several ER stress/autophagy expression markers, as well as cell survival/apoptosis markers (phospho-Akt and cleaved caspase-3) were evaluated by qPCR and/or by western blotting. As ER stress positive control, cells were treated with tunicamycin or dithiothreitol (DTT). The gene expression analyses showed a mild but significant induction of the ER stress/autophagy markers. The western blot analyses revealed that the Leishmania infection induced Akt phosphorylation and significantly inhibited the induction of caspase-3 cleavage, eIF2α phosphorylation and DDIT3/CHOP expression in tunicamycin and DTT treated cells. The mild but significant increase in ER stress expression markers and the delay/attenuation of the effects of ER stress inducers in infected cells support the hypothesis that L. infantum could promote survival of host cells by inducing a mild ER stress response. The host ER stress response could be not only a common pathogenic mechanism among Leishmania species but also a target for development of new drugs.

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“…Moreover, the diagnosis of L . infantum infection was confirmed by a real-time PCR assay from conjunctival swabs and lymph nodes [19]. …”

Section: Methodsmentioning

confidence: 99%

Leishmania infantum Induces Mild Unfolded Protein Response in Infected Macrophages

et al. 2016

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“…Recently, other body samples, such as conjunctival swabs, are being considered as a source for CL tests but will require further research [ 45 , 46 ].…”

Section: Laboratory Diagnosismentioning

confidence: 99%

Cutaneous Leishmaniasis: Recent Developments in Diagnosis and Management

2015

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This review focuses on recent developments in the diagnosis, treatment, management, and strategies for the prevention and control of cutaneous leishmaniasis (CL) caused by both Old and New World Leishmania species. CL is caused by the vector-borne protozoan parasite Leishmania and is transmitted via infected female sandflies. The disease is endemic in more than 98 countries and an estimated 350 million people are at risk. The overall prevalence is 12 million cases and the annual incidence is 2–2.5 million. The World Health Organization considers CL a severely neglected disease and a category 1 emerging and uncontrolled disease. The management of CL differs from region to region and is primarily based on local experience-based evidence. Most CL patients can be treated with topical treatments, but some Leishmania species can cause mucocutaneous involvement requiring a systemic therapeutic approach. Moreover, Leishmania species can vary in their sensitivity to available therapeutic options. This makes species determination critical for the choice of treatment and the clinical outcome of CL. Identification of the infecting parasite used to be laborious, but now the Leishmania species can be identified relatively easy with new DNA techniques that enable a more rational therapy choice. Current treatment guidelines for CL are based on poorly designed and reported trials. There is a lack of evidence for potentially beneficial treatments, a desperate need for large well-conducted studies, and standardization of future trials. Moreover, intensified research programs to improve vector control, diagnostics, and the therapeutic arsenal to contain further incidence and morbidity are needed.

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“…β-actin or beta-2-microglobulin), not only to monitor PCR inhibition (i.e. false-negative results), but also to normalize the parasite load to the amount of host cells [ 33 , 34 , 48 , 58 ]. Although some clinical samples may contain a small amount of parasite DNA, the use of high copy number sequence as targets (e.g.…”

Section: Clinical Samplesmentioning

confidence: 99%

“…We also tested the use of conjunctival swabs (CS) for the evaluation of CanL by qPCR targeting kDNA. Since we used raw CS lysates as source of Leishmania DNA, their potential inhibition in qPCR was tested: it was found that 1 μl raw lysate per 25 μl qPCR reaction mixture did not show any inhibitory effect on the assays, allowing accurate quantification [ 48 ].…”

Section: Qpcr Assays For Parasite Detection and Quantificationmentioning

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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Application of qPCR in conjunctival swab samples for the evaluation of canine leishmaniasis in borderline cases or disease relapse and correlation with clinical parameters

Cited by 5 publications

References 23 publications

Leishmania infantum Induces Mild Unfolded Protein Response in Infected Macrophages

Leishmania infantum Induces Mild Unfolded Protein Response in Infected Macrophages

Cutaneous Leishmaniasis: Recent Developments in Diagnosis and Management

Real-time PCR applications for diagnosis of leishmaniasis

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