The host micro-RNA cfa-miR-346 is induced in canine leishmaniasis.

BACKGROUND: Leishmaniases are a group of anthropo-zoonotic parasitic diseases caused by a protozoan of the Leishmania genus, affecting both humans and other vertebrates, including dogs. L. infantum is responsible for the visceral and occasionally cutaneous form of the disease in humans and canine leishmaniasis. Previously, we have shown that L. infantum induces a mild but significant increase in endoplasmic reticulum (ER) stress expression markers to promote parasites survival in human and murine infected macrophages. Moreover, we demonstrated that the miRNA hsa-miR-346, induced by the UPR-activated transcription factor sXBP1, was significantly upregulated in human macrophages infected with different L. infantum strains. However, the ER stress response in infected dogs, which represent an important reservoir for Leishmania parasite, was described once recently, whereas the miR-346 expression was not reported before. Therefore, this study aimed to investigate these pathways in the canine macrophage-like cell line DH82 infected by Leishmania spp. and to evaluate the presence of cfa-miR-346 in plasma of non-infected and infected dogs.  The DH82 cells were infected with L. infantum and L. braziliensis parasites and the expression of cfa-mir-346 and several ER stress markers was evaluated by quantitative PCR (qPCR) at different time points. Furthermore, the cfa-miR-346 was monitored in plasma collected from non-infected dogs (n = 11) and dogs naturally infected by L. infantum (n = 18). RESULTS: The results in DH82 cells showed that cfa-mir-346 was induced at both 24 h and 48 h post-infection with all Leishmania strains but not with tunicamycin, accounting for a mechanism of induction independent from sXBP1, unlike what was previously observed in human cell lines. Moreover, the cfa-miR-346 expression analysis on plasma revealed a significant increase in infected dogs compared to non-infected dogs. CONCLUSIONS: Here for the first time, we report the upregulation of cfa-miR-346 induced by Leishmania infection in canine macrophage-like cells and plasma samples of naturally infected dogs. According to our results, the cfa-miR-346 appears to be linked to infection, and understanding its role and identifying its target genes could contribute to elucidate the mechanisms underlying the host-pathogen interaction in leishmaniasis.

The use of conjunctival swabs in the diagnosis of human visceral leishmaniasis.

Human visceral leishmaniasis (VL) is a severe disease whose diagnosis comprises immunological tests, microscopic biopsy examination, and biomolecular assays. In veterinary medicine, conjunctival swabs are widely used for detection of parasite DNA. Here, we describe the case of human VL in which conjunctival swabs were successfully used for Leishmania detection.

Transcriptional signatures in human macrophage-like cells infected by Leishmania infantum, Leishmania major and Leishmania tropica.

BACKGROUND: In the Mediterranean basin, three Leishmania species have been identified: L. infantum, L. major and L. tropica, causing zoonotic visceral leishmaniasis (VL), zoonotic cutaneous leishmaniasis (CL) and anthroponotic CL, respectively. Despite animal models and genomic/transcriptomic studies provided important insights, the pathogenic determinants modulating the development of VL and CL are still poorly understood. This work aimed to identify host transcriptional signatures shared by cells infected with L. infantum, L. major, and L. tropica, as well as specific transcriptional signatures elicited by parasites causing VL (i.e., L. infantum) and parasites involved in CL (i.e., L. major, L. tropica). METHODOLOGY/PRINCIPAL FINDINGS: U937 cells differentiated into macrophage-like cells were infected with L. infantum, L. major and L. tropica for 24h and 48h, and total RNA was extracted. RNA sequencing, performed on an Illumina NovaSeq 6000 platform, was used to evaluate the transcriptional signatures of infected cells with respect to non-infected cells at both time points. The EdgeR package was used to identify differentially expressed genes (fold change > 2 and FDR-adjusted p-values < 0.05). Then, functional enrichment analysis was employed to identify the enriched ontology terms in which these genes are involved. At 24h post-infection, a common signature of 463 dysregulated genes shared among all infection conditions was recognized, while at 48h post-infection the common signature was reduced to 120 genes. Aside from a common transcriptional response, we evidenced different upregulated functional pathways characterizing L. infantum-infected cells, such as VEGFA-VEGFR2 and NFE2L2-related pathways, indicating vascular remodeling and reduction of oxidative stress as potentially important factors for visceralization. CONCLUSIONS: The identification of pathways elicited by parasites causing VL or CL could lead to new therapeutic strategies for leishmaniasis, combining the canonical anti-leishmania compounds with host-directed therapy.

High-resolution melting (HRM)-based detection of polymorphisms in the malic enzyme and glucose-6-phosphate isomerase genes for Leishmania infantum genotyping.

BACKGROUND: Leishmaniasis is a zoonotic disease endemic in the Mediterranean region where Leishmania infantum is the causative agent of human and canine infection. Characterization of this parasite at the subspecies level can be useful in epidemiological studies, to evaluate the clinical course of the disease (e.g. resistant strains, visceral and cutaneous forms of leishmaniasis) as well as to identify infection reservoirs. Multilocus enzyme electrophoresis (MLEE), a method currently recognized as the reference method for characterizing and identifying strains of Leishmania, is cumbersome and time-consuming and requires cultured parasites. These disadvantages have led to the development of other methods, such as multilocus microsatellite typing (MLMT) and multilocus sequence typing (MLST), for typing Leishmania parasites; however, these methods have not yet been applied for routine use. In this study, we first used MLST to identify informative polymorphisms on single-copy genes coding for metabolic enzymes, following which we developed two rapid genotyping assays based on high-resolution melting (HRM) analysis to explore these polymorphisms in L. infantum parasites. METHODS: A customized sequencing panel targeting 14 housekeeping genes was designed and MLST analysis was performed on nine L. infantum canine and human strains/isolates. Two quantitative real-time PCR-HRM assays were designed to analyze two informative polymorphisms on malic enzyme (ME) and glucose-6-phosphate isomerase (GPI) genes (390T/G and 1831A/G, respectively). The two assays were applied to 73 clinical samples/isolates from central/southern Italy and Pantelleria island, and the results were confirmed by DNA sequencing in a subset of samples. RESULTS: The MLST analysis, together with sequences available in the Genbank database, enabled the identification of two informative polymorphisms on the genes coding for ME and GPI. The fast screening of these polymorphisms using two HRM-based assays in 73 clinical samples/isolates resulted in the identification of seven genotypes. Overall, genotype 1 (sequence type 390T/1831G) was the most highly represented (45.2%) in the overall sample and correlated with the most common L. infantum zymodemes (MON-1, MON-72). Interestingly, in Pantelleria island, the most prevalent genotype (70.6%) was genotype 6 (sequence type 390T/1831A). CONCLUSIONS: Applying our HRM assays on clinical samples allowed us to identify seven different genotypes without the need for parasite isolation and cultivation. We have demonstrated that these assays could be used as fast, routine and inexpensive tools for epidemiological surveillance of L. infantum or for the identification of new infection reservoirs.

Rapid monitoring of SARS-CoV-2 variants of concern through high-resolution melt analysis.

The current global pandemic of COVID-19 is characterized by waves of infection due to the emergence of new SARS-CoV-2 variants carrying mutations on the Spike (S) protein gene. Since autumn 2020 many Variants of Concern (VOC) have been reported: Alpha/B.1.1.7, Beta/B.1.351, Gamma/P.1, Delta/B.1.617.2, Omicron/B.1.1.529, and sublineages. Surveillance of genomic variants is currently based on whole-genome sequencing (WGS) of viral genomes on a random fraction of samples positive to molecular tests. WGS involves high costs, extended analysis time, specialized staff, and expensive instruments compared to a PCR-based test. To rapidly identify the VOCs in positive samples, six assays based on real-time PCR and high-resolution melting (HRM) were designed on the S gene and applied to 120 oro/nasopharyngeal swab samples collected from October 2020 to June 2022 (106 positive and 14 negative samples). Overall, the assays showed 100% specificity and sensitivity compared with commercial PCR tests for COVID-19. Moreover, 104 samples out of 106 (98.1%) were correctly identified as follows: 8 Wuhan (wild type), 12 Alpha, 23 Delta, 46 Omicron BA.1/BA.1.1, 15 Omicron BA.2/BA.4/BA.5. With our lab equipment, about 10 samples can be processed every 3 h at the cost of less than € 10 ($ 10.60) per sample, including RNA extraction. The implementation of this approach could help local epidemiological surveillance and clinical decision-making.

Comparing Heterologous and Homologous COVID-19 Vaccination: A Longitudinal Study of Antibody Decay.

The humoral response after vaccination was evaluated in 1248 individuals who received different COVID-19 vaccine schedules. The study compared subjects primed with adenoviral ChAdOx1-S (ChAd) and boosted with BNT162b2 (BNT) mRNA vaccines (ChAd/BNT) to homologous dosing with BNT/BNT or ChAd/ChAd vaccines. Serum samples were collected at two, four and six months after vaccination, and anti-Spike IgG responses were determined. The heterologous vaccination induced a more robust immune response than the two homologous vaccinations. ChAd/BNT induced a stronger immune response than ChAd/ChAd at all time points, whereas the differences between ChAd/BNT and BNT/BNT decreased over time and were not significant at six months. Furthermore, the kinetic parameters associated with IgG decay were estimated by applying a first-order kinetics equation. ChAd/BNT vaccination was associated with the longest time of anti-S IgG negativization and with a slow decay of the titer over time. Finally, analyzing factors influencing the immune response by ANCOVA analysis, it was found that the vaccine schedule had a significant impact on both the IgG titer and kinetic parameters, and having a Body Mass Index (BMI) above the overweight threshold was associated with an impaired immune response. Overall, the heterologous ChAd/BNT vaccination may offer longer-lasting protection against SARS-CoV-2 than homologous vaccination strategies.

Evaluation of Two-Month Antibody Levels after Heterologous ChAdOx1-S/BNT162b2 Vaccination Compared to Homologous ChAdOx1-S or BNT162b2 Vaccination.

We evaluated the post-vaccination humoral response of three real-world cohorts. Vaccinated subjects primed with ChAdOx1-S and boosted with BNT162b2 mRNA vaccine were compared to homologous dosing (BNT162b2/BNT162b2 and ChAdOx1-S/ChAdOx1-S). Serum samples were collected two months after vaccination from a total of 1248 subjects. The results showed that the heterologous vaccine schedule induced a significantly higher humoral response followed by homologous BNT162b2/BNT162b2 and ChAdOx1-S/ChAdOx1-S vaccines (p < 0.0001). Moreover, analyzing factors (i.e., vaccine schedule, sex, age, BMI, smoking, diabetes, cardiovascular diseases, respiratory tract diseases, COVID-19 diagnosis, vaccine side effects) influencing the IgG anti-S response, we found that only the type of vaccine affected the antibody titer (p < 0.0001). Only mild vaccine reactions resolved within few days (40% of subjects) and no severe side effects for either homologous groups or the heterologous group were reported. Our data support the use of heterologous vaccination as an effective and safe alternative to increase humoral immunity against COVID-19.

In Vitro Reduced Susceptibility to Pentavalent Antimonials of a Leishmania infantum Isolate from a Human Cutaneous Leishmaniasis Case in Central Italy.

Cutaneous leishmaniasis (CL) caused by Leishmania (Leishmania) infantum is endemic in the Mediterranean basin. Here we report an autochthonous case of CL in a patient living in central Italy with an unsatisfactory response to treatment with intralesional Meglumine Antimoniate and in vitro demonstration of reduced susceptibility to SbIII. Parasitological diagnosis was first achieved by histopathology on tissue biopsy and the patient was treated with a local infiltration of Meglumine Antimoniate. Since the clinical response at 12 weeks from the treatment's onset was deemed unsatisfactory, two further skin biopsies were taken for histopathological examination, DNA extraction and parasite isolation. L. (L.) infantum was identified by molecular typing. The low susceptibility to Meglumine Antimoniate was confirmed in vitro: the promastigotes from the patient strain showed significantly lower susceptibility to SbIII (the active trivalent form of antimonial) compared to the reference strain MHOM/TN/80/IPT1. The patient underwent a new treatment course with intravenous liposomal Amphotericin B, reaching complete healing of the lesion. Additional studies are needed to confirm the epidemiological and clinical relevance of reduced susceptibility to SbIII of human L. (L.) infantum isolate in Italy.

Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World Leishmania Species.

The parasite protozoan Leishmania, the causative agent of leishmaniasis, includes two subgenera of medical interest: Leishmania (Leishmania) and Leishmania (Viannia). Parasite species detection and characterization is crucial to choose treatment protocols and to monitor the disease evolution. Molecular approaches can speed up and simplify the diagnostic process. In particular, several molecular assays target the mitochondrial DNA minicircle network (kDNA) that characterizes the Leishmania genus. We previously proposed a qPCR assay targeting kDNA, followed by high resolution melt (HRM) analysis (qPCR-ML) to distinguish L. (L.) infantum and L. (L.) amazonensis from L. Viannia species. Successively, this assay has been integrated with other qPCR assays, to differentiate L. (L.) infantum, L. (L.) amazonensis and L. (L.) mexicana. In this work, we tested the applicability of our qPCR-ML assay on L. (L.) donovani, L. (L.) major, L. (L.) tropica and L. (L.) aethiopica, showing that the qPCR-ML assay can also amplify Old World species, different from L. (L.) infantum, with good quantification limits (1 × 10-4-1 × 10-6 ng/pcr tube). Moreover, we evaluated 11 L. (L.) infantum strains/isolates, evidencing the variability of the kDNA minicircle target molecules among the strains/isolates of the same species, and pointing out the possibility of quantification using different strains as reference. Taken together, these data account for the consideration of qPCR-ML as a quantitative pan-Leishmania assay.

Real-time PCR to differentiate among Leishmania (Viannia) subgenus, Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis: Application on Brazilian clinical samples.

Leishmaniasis is a complex disease caused by Leishmania species belonging to subgenera Leishmania and Viannia. In South America, L. (L.) infantum is considered the most important causative agent of visceral leishmaniasis, while L. (L.) amazonensis and Viannia subgenus species are responsible for the different cutaneous or mucocutaneous forms. In our previous work, we developed a diagnostic approach for Leishmania species discrimination based on two qPCRs (qPCR-ML and qPCR-ama) targeting the minicircle kDNA followed by melting analysis. This approach allowed to (i) differentiate the subgenera Leishmania and Viannia, and (ii) distinguish between L. (L.) infantum and L. (L.) amazonensis. The aim of this work was to demonstrate the applicability of the approach previously described, using human and canine clinical samples and strains from a Brazilian region, where L. (L.) infantum, L. (L.) amazonensis and Viannia subgenus species coexist. After validation on New World strains, the diagnostic approach was applied blindly to 36 canine clinical samples (peripheral blood and bone marrow) and 11 human clinical samples (peripheral blood and bone marrow). The sensitivity was 95.6% (95% confidence interval 77.3-100%) and 100% (95% confidence interval 76.9-100%) in the canine bone marrow samples and human (peripheral blood and bone marrow) samples, respectively, compared to conventional PCR assays. Concerning the Leishmania species identification, the conventional and qPCR-based methods showed kappa value of 0.876 (95% confidence interval 0.638-1.000), indicating good agreement. Therefore, this approach proved to be useful in both veterinary and human clinical context in regions co-endemic for L. (L.) infantum, L. (L.) amazonensis, and Viannia subgenus, helping to provide rapid diagnosis and to allow studies of species distribution.

Differentiation of Leishmania (L.) infantum, Leishmania (L.) amazonensis and Leishmania (L.) mexicana Using Sequential qPCR Assays and High-Resolution Melt Analysis.

Leishmania protozoa are the etiological agents of visceral, cutaneous and mucocutaneous leishmaniasis. In specific geographical regions, such as Latin America, several Leishmania species are endemic and simultaneously present; therefore, a diagnostic method for species discrimination is warranted. In this attempt, many qPCR-based assays have been developed. Recently, we have shown that L. (L.) infantum and L. (L.) amazonensis can be distinguished through the comparison of the Cq values from two qPCR assays (qPCR-ML and qPCR-ama), designed to amplify kDNA minicircle subclasses more represented in L. (L.) infantum and L. (L.) amazonensis, respectively. This paper describes the application of this approach to L. (L.) mexicana and introduces a new qPCR-ITS1 assay followed by high-resolution melt (HRM) analysis to differentiate this species from L. (L.) amazonensis. We show that L. (L.) mexicana can be distinguished from L. (L.) infantum using the same approach we had previously validated for L. (L.) amazonensis. Moreover, it was also possible to reliably discriminate L. (L.) mexicana from L. (L.) amazonensis by using qPCR-ITS1 followed by an HRM analysis. Therefore, a diagnostic algorithm based on sequential qPCR assays coupled with HRM analysis was established to identify/differentiate L. (L.) infantum, L. (L.) amazonensis, L. (L.) mexicana and Viannia subgenus. These findings update and extend previous data published by our research group, providing an additional diagnostic tool in endemic areas with co-existing species.

Data on the differentiation among Leishmania (Viannia) spp., Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis in Brazilian clinical samples using real-time PCR.

This article contains the data regarding Leishmania species identification in human and canine clinical samples from a Brazilian region endemic for Leishmania (Viannia) spp., Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis, using a previously developed approach involving two qPCR assays (qPCR-ML and qPCR-ama). The data are related to the article "Real-time PCR to differentiate among Leishmania (Viannia) subgenus, Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis: application on Brazilian clinical samples" [1], and include also details of clinical evaluation/diagnosis of human patients and primer sequences used in the qPCR assays. The Leishmania species has been determined in 27 canine samples and 11 human samples, exploiting HRM analysis of qPCR-ML and Cq values of qPCR-ML and qPCR-ama, as reported previously [2]. The qPCR data were in agreement with the species characterization obtained with other methods such as conventional species-specific PCR, ITS1 PCR-RFLP or DNA sequencing. Despite the limited number of clinical samples, these data are encouraging for a potential application in regions where L. (Viannia) spp., L. (L.) infantum and L. (L.) amazonensis are co-endemic.

Exploiting genetic polymorphisms in metabolic enzymes for rapid screening of Leishmania infantum genotypes.

BACKGROUND: Leishmania infantum is the aetiological agent of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Numerous strains and/or zymodemes have been identified and characterized by multilocus enzyme electrophoresis (MLEE). MLEE is considered the reference method for L. infantum parasite typing and it is based upon enzyme electrophoretic mobility analysis from promastigote cultures. However, the MLEE technique is cumbersome, time-consuming and does not detect silent genetic mutations or nucleotide changes that give rise to amino acid changes that do not alter electrophoretic mobility. As a result of these difficulties, many DNA-based typing methods have been developed over the past few years. However, relative to the enzymes utilized in MLEE analysis, we observed a shortage of DNA sequences available in the GenBank database or an absolute lack of sequences belonging to specific zymodemes. The aims of the present study were to (i) implement the number of sequences coding for metabolic enzymes used in MLEE; (ii) identify polymorphisms that characterize L. infantum zymodemes most prevalent in the Mediterranean basin; and (iii) exploit these polymorphisms to develop a rapid screening test that would give results comparable with existing MLEE typing. RESULTS: Partial sequences of seven metabolic enzyme genes (malic enzyme, 6-phosphogluconate dehydrogenase, mitochondrial isocitrate dehydrogenase, glucose-6-phosphate isomerase, glucose-6-phosphate dehydrogenase, phosphoglucomutase and mannose phosphate isomerase) were obtained from 11 L. infantum strains. The comparison of these sequences with those obtained from GenBank allowed for the identification of a few polymorphisms that could distinguish several zymodemes. In particular, the polymorphism 390T>G in the malic enzyme gene has been exploited to develop a high-resolution melt (HRM)-based assay to rapidly differentiate the genotype 390T, associated with zymodemes MON-1, MON-72 and MON-201, evidencing a partial agreement between genotyping results and MLEE. The assay has been successfully applied to L. infantum clinical isolates and clinical samples. CONCLUSIONS: A HRM-based assay for rapid identification of genotypes associated with the most common L. infantum zymodemes in the Mediterranean basin has been developed and its potential application in epidemiological research for L. infantum population screening, without parasite isolation and culturing, has been demonstrated.

Real-time PCR applications for diagnosis of leishmaniasis.

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.

Leishmania Infection Induces MicroRNA hsa-miR-346 in Human Cell Line-Derived Macrophages.

Leishmaniasis is an anthropo-zoonotic disease caused by various Leishmania species. The clinical manifestations of the disease vary according to the species and host characteristics. Leishmania infection leads to subversion/modulation of the host's innate immune response and cellular metabolic pathways. In the last years, it has been shown that many host cell gene expression and signaling pathways are targeted by Leishmania to subvert host defenses (e.g., oxidative damage, immune activation, antigen presentation, apoptosis) and allow parasite survival and replication. However, the molecular mechanisms triggered by the parasite are not fully elucidated. The role of miRNA has recently been evaluated in human or murine macrophages infected with Leishmania (Leishmania) major, L. (L.) donovani or L. (L.) amazonensis. However, no literature exists regarding miRNA dysregulation in host cells infected with L. (L.) infantum or L. (Viannia) species. Since we previously showed that L. (L.) infantum infection induced unfolded protein response (UPR) in macrophages, we focused on miR-346, which has been shown to be induced by the UPR-activated transcription factor sXBP1 and has a potential role in the modulation of the immune response. Macrophages differentiated from U937 and/or THP-1 human monocytic cells were infected with four L. (L.) infantum strain/clinical isolates and one L. (V.) sp. clinical isolate. A significant upregulation of miR-346 (p < 0.05) was observed in infections with all the Leishmania species tested. Moreover, RFX1 (a miR-346 predicted target gene) was found to be significantly downregulated (p < 0.05) after 48h infection, and miR-346 was found to have a role in this downregulation. The induction of miR-346 in macrophages infected with L. (L.) infantum and L. (V.) sp., reported here for the first time, could play a role in regulating macrophage functions since several MHC- or interferon-associated genes are among the targets of this miRNA. Hence, miR-346 could be considered an attractive anti-Leishmania drug target.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Leishmania infantum Induces Mild Unfolded Protein Response in Infected Macrophages.

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.

Detection and characterization of Leishmania (Leishmania) and Leishmania (Viannia) by SYBR green-based real-time PCR and high resolution melt analysis targeting kinetoplast minicircle DNA.

Leishmaniasis is a neglected disease with a broad clinical spectrum which includes asymptomatic infection. A thorough diagnosis, able to distinguish and quantify Leishmania parasites in a clinical sample, constitutes a key step in choosing an appropriate therapy, making an accurate prognosis and performing epidemiological studies. Several molecular techniques have been shown to be effective in the diagnosis of leishmaniasis. In particular, a number of PCR methods have been developed on various target DNA sequences including kinetoplast minicircle constant regions. The first aim of this study was to develop a SYBR green-based qPCR assay for Leishmania (Leishmania) infantum detection and quantification, using kinetoplast minicircle constant region as target. To this end, two assays were compared: the first used previously published primer pairs (qPCR1), whereas the second used a nested primer pairs generating a shorter PCR product (qPCR2). The second aim of this study was to evaluate the possibility to discriminate among subgenera Leishmania (Leishmania) and Leishmania (Viannia) using the qPCR2 assay followed by melting or High Resolution Melt (HRM) analysis. Both assays used in this study showed good sensitivity and specificity, and a good correlation with standard IFAT methods in 62 canine clinical samples. However, the qPCR2 assay allowed to discriminate between Leishmania (Leishmania) and Leishmania (Viannia) subgenera through melting or HRM analysis. In addition to developing assays, we investigated the number and genetic variability of kinetoplast minicircles in the Leishmania (L.) infantum WHO international reference strain (MHOM/TN/80/IPT1), highlighting the presence of minicircle subclasses and sequence heterogeneity. Specifically, the kinetoplast minicircle number per cell was estimated to be 26,566±1,192, while the subclass of minicircles amplifiable by qPCR2 was estimated to be 1,263±115. This heterogeneity, also observed in canine clinical samples, must be taken into account in quantitative PCR-based applications; however, it might also be used to differentiate between Leishmania subgenera.

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

BACKGROUND: In leishmaniasis caused by Leishmania infantum, the dog acts as the main reservoir for the disease. Non-invasive sampling for Leishmania detection is pivotal for rapid and affordable diagnosis. Recently, the use of conjunctival swab (CS) has been evaluated as a non-invasive sampling technique for quantitative real-time PCR (qPCR). However, few investigations have been made on the applicability of CS qPCR in particular cases such as dogs with borderline IFAT titres, suspected disease relapse with comorbidity and therapy monitoring. The aims of this study were i) to confirm the efficacy of CS, comparing these samples to buffy coat (BC) samples, as effective non-invasive samples for Leishmania quantitative detection by qPCR and ii) to verify the usefulness of qPCR compared to conventional laboratory and clinical parameters to assist in therapeutic decision making regarding dogs with complex clinical cases. METHODS: Eighty dogs were divided into 4 groups based on their IFAT titres and clinical histories. Two qPCR assays were performed both on CS raw lysates and on purified DNA from BC samples. The assays were then compared. Z tests for difference of proportion, with Bonferroni correction, were carried out to evaluate the qPCR results. Logistic regression with backward stepwise elimination was performed to detect the subset of haematochemical variables significantly associated with PCR positivity. RESULTS: The qPCR performed on CS samples showed better sensitivity (87%) and specificity (96%) than assays carried out using BC samples, regardless of the primers used. The haematochemical parameters haemoglobin and globulins were found to be significantly associated with qPCR positivity. Pearson correlations between Leishmania kDNA load in CS and body condition scores or IFAT titres were calculated in dogs with new leishmaniasis diagnoses. The Leishmania kDNA load in CS correlated moderately with IFAT titres (R = 0.59) but a very weak correlation (R = 0.37) with body condition score (BCS) was found. CONCLUSIONS: The applicability of CS for Leishmania detection in dogs was confirmed, revealing the usefulness of raw lysates for quantitative purposes. Moreover, the qPCR was found to be particularly useful in cases lacking a clear clinical diagnosis, where the haematochemical values cannot be predictive.