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.

Autophagy is a cellular mechanism by which cells degrade intracellular components in lysosomes, maintaining cellular homeostasis. It has been hypothesized that autophagy could have a role in cancer prevention through the elimination of damaged proteins and organelles; this could explain epidemiological evidence showing the chemopreventive properties of the autophagy-inducer metformin. In this study, we analyzed the autophagy-related effect of metformin in both cancer initiation and progression in non-tumorigenic cells. We also analyzed the induction of tumorigenesis in autophagy-deficient cells, and its correlation with the ER stress. Our results showed that metformin induced massive cell death in preneoplastic JB6 Cl 41-5a cells treated with tumor promoter (phorbol) and in NIH/3T3 treated with H₂O₂. Inhibiting autophagy with wortmannin or ATG7 silencing, the effect of metformin decreased, indicating an autophagy-related cytotoxic activity under stress conditions. We also found an induction of tumorigenesis in ATG7-silenced NIH/3T3 cell clone (3T3-619C3 cells), but not in wild-type and in scrambled transfected cells, and an upregulation of unfolded protein response (UPR) markers in 3T3-619C3 cells treated with H₂O₂. These findings suggest that autophagic cell death could be considered as a new mechanism by which eliminate damaged cells, representing an attractive strategy to eliminate potential tumorigenic cells.

Perturbations of the physiological status of the endoplasmic reticulum (ER) trigger a specific response known as the ER stress response or unfolded protein response (UPR). In mammalian cells, the UPR is mediated by three ER transmembrane proteins (IRE1, PERK and ATF6) which activate three signaling cascades to restore ER homeostasis. In recent years, a cross-talk between UPR, inflammatory and microbial sensing pathways has been elucidated. Pathogen infection can lead to UPR activation; moreover, several pathogens subvert the UPR to promote their survival and replication. While the UPR in viral and bacterial infection has been characterized, little is known about the role of UPR in intracellular parasite infection. Here, we review recent findings on UPR induction/modulation by intracellular parasites in host cells. Lay abstract The endoplasmic reticulum (ER) has a central role in maintaining homeostasis and in the regulation of innate immune response. Perturbations in the ER (ER stress) lead to a signaling cascade termed unfolded protein response (UPR), aimed at restoring cell homeostasis. The UPR pathways are strictly connected with innate immunity and inflammation. In fact, many pathogens (mainly viruses and bacteria) are known to induce/modulate the UPR in the host cell. The UPR triggered by intracellular parasites is still poorly investigated. Its characterization could contribute to explain the mechanisms of pathogenicity and to identify targets for the development of new therapeutic approaches.

Preimplantation genetic diagnosis (PGD) currently relies on biopsy of one or few embryo cells. Our aim was to evaluate the embryo extracellular matrices (spent medium and blastocoele fluid) as source of DNA for embryo genotyping. We first evaluated the amplifiability and the amount of genomic DNA in spent embryo culture media from day 3 (n = 32) and day 5/6 (n = 54). Secondly, we evaluated the possibility to genotype the MTHFR polymorphism C677T from media at day 5/6 (n = 8) and blastocoele fluids (n = 9) by direct sequencing. The C677T polymorphism detection rate was 62.5 and 44.4% in medium and fluid, respectively. A noninvasive approach for embryo genotyping was possible, but still with limitations due to low detection rate and possible allele dropout.

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.

Leishmaniasis 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. DNA 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. The 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. A 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.