Leishmania amazonensis infection regulates oxidate stress in hyperglycemia and diabetes impairing macrophage's function and immune response.

Leishmaniasis is a group of infectious diseases caused by protozoa of the Leishmania genus and its immunopathogenesis results from an unbalanced immune response during the infection. Diabetes is a chronic disease resulting from dysfunction of the body's production of insulin or the ability to use it properly, leading to hyperglycemia causing tissue damage and impairing the immune system. AIMS: The objective of this work was to evaluate the effects of hyperglycemia and diabetes during Leishmania amazonensis infection and how these conditions alter the immune response to the parasite. METHODS: An in vitro hyperglycemic stimulus model using THP-1-derived macrophages and an in vivo experimental diabetes with streptozotocin (STZ) in C57BL/6 mice was employed to investigate the impact of diabetes and hyperglicemia in Leishmania amazonensis infection. RESULTS: We observed that hyperglycemia impair the leishmanicidal capacity of macrophages derived from THP-1 cells and reverse the resistance profile that C57BL/6 mice have against infection by L. amazonensis, inducing more exacerbated lesions compared to non-diabetic animals. In addition, the hyperglycemic stimulus favored the increase of markers related to the phenotype of M2 macrophages. The induction of experimental diabetes in C57BL/6 mice resulted in a failure in the production of nitric oxide (NO) in the face of infection and macrophages from diabetic animals failed to process and present Leishmania antigens, being unable to activate and induce proliferation of antigen-specific lymphocytes. CONCLUSION: Together, these data demonstrate that diabetes and hyperglycemia can impair the cellular immune response, mainly of macrophages, against infection by parasites of the genus Leishmania.

Swarna Bhasma Induces Antigen-Presenting Abilities of Macrophages and Helps Antigen Experienced CD4+ T Cells to Acquire Th1 Phenotypes Against Leishmania donovani Antigens.

In leishmaniasis, the protective immunity is largely mediated by proinflammatory cytokine producing abilities of T cells and an efficient parasite killing by phagocytic cells. Notwithstanding a substantial progress that has been made during last decades, the mechanisms or factors involved in establishing protective immunity against Leishmania are not identified. In ancient Indian literature, metallic "bhasma," particularly that of "swarna" or gold (fine gold particles), is indicated as one of the most prominent metal-based therapeutic medicine, which is known to impart protective and curative properties in various health issues. In this work, we elucidated the potential of swarna bhasma (SB) on the effector properties of phagocytes and antigen-activated CD4+ T cells in augmenting the immunogenicity of L. donovani antigens. The characterization of SB revealing its shape, size, composition, and measurement of cytotoxicity established the physiochemical potential for its utilization as an immunomodulator. The activation of macrophages with SB enhanced their capacity to produce nitric oxide and proinflammatory cytokines, which eventually resulted in reduced uptake of parasites and their proliferation in infected cells. Further, in Leishmania-infected animals, SB administration reduced the generation of IL-10, an anti-inflammatory cytokine, and enhanced pro-inflammatory cytokine generation by antigen activated CD4+ T cells with increased frequency of double (IFNγ+/TNFα+) and triple (IFNγ+TNFα+IL-2+) positive cells and abrogated disease pathogeneses at the early days of infection. Our results also suggested that cow-ghee (A2) emulsified preparation of SB, either alone or with yashtimadhu, a known natural immune modulator which enhances the SB's potential in enhancing the immunogenicity of parasitic antigens. These findings suggested a definite potential of SB in enhancing the effector functions of phagocytes and CD4+ T cells against L. donovani antigens. Therefore, more studies are needed to elucidate the mechanistic details of SB and its potential in enhancing vaccine-induced immunity.

The potential role of protein disulfide isomerases (PDIs) during parasitic infections: a focus on Leishmania spp.

Leishmaniasis is a group of vector-borne diseases caused by intracellular protozoan parasites belonging to the genus Leishmania. Leishmania parasites can employ different and numerous sophisticated strategies, including modulating host proteins, cell signaling, and cell responses by parasite proteins, to change the infected host conditions to favor the parasite persistence and induce pathogenesis. In this sense, protein disulfide isomerases (PDIs) have been described as crucial proteins that can be modulated during leishmaniasis and affect the pathogenesis process. The effect of modulated PDIs can be investigated in both aspects, parasite PDIs and infected host cell PDIs, during infection. The information concerning PDIs is not sufficient in parasitology; however, this study aimed to provide data regarding the biological functions of such crucial proteins in parasites with a focus on Leishmania spp. and their relevant effects on the pathogenesis process. Although there are no clinical trial vaccines and therapeutic approaches, highlighting this information might be fruitful for the development of novel strategies based on PDIs for the management of parasitic diseases, especially leishmaniasis.

Interactions between Leishmania parasite and sandfly: a review.

Leishmaniasis transmission cycles are maintained and sustained in nature by the complex crosstalk of the Leishmania parasite, sandfly vector, and the mammalian hosts (human, as well as zoonotic reservoirs). Regardless of the vast research on human host-parasite interaction, there persists a substantial knowledge gap on the parasite's development and modulation in the vector component. This review focuses on some of the intriguing aspects of the Leishmania-sandfly interface, beginning with the uptake of the intracellular amastigotes from an infected host to the development of the parasite within the sandfly's alimentary canal, followed by the transmission of infective metacyclic stages to another potential host. Upon ingestion of the parasite, the sandfly hosts an intricate repertoire of immune barriers, either to evade the parasite or to ensure its homeostatic coexistence with the vector gut microbiome. Sandfly salivary polypeptides and Leishmania exosomes are co-egested with the parasite inoculum during the infected vector bite. This has been attributed to the modulation of the parasite infection and subsequent clinical manifestation in the host. While human host-based studies strive to develop effective therapeutics, a greater understanding of the vector-parasite-microbiome and human host interactions could help us to identify the targets and to develop strategies for effectively preventing the transmission of leishmaniasis.

The immune response in canine and human leishmaniasis and how this influences the diagnosis- a review and assessment of recent research.

Leishmaniasis is a widespread but still underdiagnosed parasitic disease that affects both humans and animals. There are at least 20 pathogenic species of Leishmania, most of them being zoonotic. The diagnosis of leishmaniasis remains a major challenge, with an important role being played by the species of parasites involved, the genetic background, the immunocompetence of the host. This paper brings to the fore the sensitivity of the balance in canine and human leishmaniasis and addresses the importance of the host's immune response in establishing a correct diagnosis, especially in certain cases of asymptomatic leishmaniasis, or in the situation the host is immunosuppressed or acquired leishmaniasis through vertical transmission. The methods considered as a reference in the diagnosis of leishmaniasis no longer present certainty, the diagnosis being influenced mostly by the immune response of the host, which differs according to the presence of other associated diseases or even according to the breed in dogs. Consequently, the diagnosis and surveillance of leishmaniasis cases remains an open topic, requiring new diagnostic methods adapted to the immunological state of the host.

LeishMANIAdb: a comparative resource for Leishmania proteins.

Leishmaniasis is a detrimental disease causing serious changes in quality of life and some forms can lead to death. The disease is spread by the parasite Leishmania transmitted by sandfly vectors and their primary hosts are vertebrates including humans. The pathogen penetrates host cells and secretes proteins (the secretome) to repurpose cells for pathogen growth and to alter cell signaling via host-pathogen protein-protein interactions). Here, we present LeishMANIAdb, a database specifically designed to investigate how Leishmania virulence factors may interfere with host proteins. Since the secretomes of different Leishmania species are only partially characterized, we collated various experimental evidence and used computational predictions to identify Leishmania secreted proteins to generate a user-friendly unified web resource allowing users to access all information available on experimental and predicted secretomes. In addition, we manually annotated host-pathogen interactions of 211 proteins and the localization/function of 3764 transmembrane (TM) proteins of different Leishmania species. We also enriched all proteins with automatic structural and functional predictions that can provide new insights in the molecular mechanisms of infection. Our database may provide novel insights into Leishmania host-pathogen interactions and help to identify new therapeutic targets for this neglected disease. Database URL  https://leishmaniadb.ttk.hu/.

In Silico Exploration of the Trypanothione Reductase (TryR) of L. mexicana.

Human leishmaniasis is a neglected tropical disease which affects nearly 1.5 million people every year, with Mexico being an important endemic region. One of the major defense mechanisms of these parasites is based in the polyamine metabolic pathway, as it provides the necessary compounds for its survival. Among the enzymes in this route, trypanothione reductase (TryR), an oxidoreductase enzyme, is crucial for the Leishmania genus' survival against oxidative stress. Thus, it poses as an attractive drug target, yet due to the size and features of its catalytic pocket, modeling techniques such as molecular docking focusing on that region is not convenient. Herein, we present a computational study using several structure-based approaches to assess the druggability of TryR from L. mexicana, the predominant Leishmania species in Mexico, beyond its catalytic site. Using this consensus methodology, three relevant pockets were found, of which the one we call σ-site promises to be the most favorable one. These findings may help the design of new drugs of trypanothione-related diseases.

Cellular mediators in human leishmaniasis: Critical determinants in parasite killing or disease progression.

Data on cellular immunity mediators in the early phase of human leishmaniasis are still limited and controversial. In order to mimic the changes of humoral mediators during the early phase of human natural infection, some Th1, Th2, Treg, and Breg cytokines, MCP-1, and the nitric oxide (NO) from human PBMC, stimulated by Leishmania infantum, Leishmania major, Leishmania donovani and Leishmania tropica infective metacyclic promastigotes, were determined. After 4 h of L. major, L. donovani, and L. tropica challenge, TNFα, IL-1ß, IL-6 levels were significantly higher than negative control cultures with saline (SF) instead of Leishmania promastigotes, unlike L. infantum-stimulated TNFα and L. major-stimulated IL-1ß. We obtained higher levels of IL-4 and IL-10 cytokines after stimulation of human PBMCs by L. infantum and L. donovani, compared to those observed after the challenge of PBMCs by L. major and L. tropica. Regarding IL-35, such cytokine levels were significantly increased following infection with L. infantum and L. donovani, in contrast to L. major and L. tropica. Up to our knowledge, we are the first to study the effect of four different species of Leishmania on IL-35 levels in human cells. Our study highlights how several Leishmania species can up-regulate different groups of cytokines (Th1, Th2, Treg and Breg) and modulate NO release in a different way. This original aspect can be explained by different Leishmania cell products, such as LPG, obtained from different strains/species of live parasites. Our findings would contribute to the development of new therapeutics or vaccination strategies.

Access and utilization of host-derived iron by Leishmania parasites.

Iron is involved in many biochemical processes including oxygen transport, ATP production, DNA synthesis and antioxidant defense. The importance of iron also applies to Leishmania parasites, an intracellular protozoan pathogen causing leishmaniasis. Leishmania are heme-auxotrophs, devoid of iron storage proteins and the heme synthesis pathway. Acquisition of iron and heme from the surrounding niche is thus critical for the intracellular survival of Leishmania inside the host macrophages. Moreover, Leishmania parasites are also exposed to oxidative stress within phagolysosomes of macrophages in mammalian hosts, and they need iron superoxide dismutase for overcoming this stress. Therefore, untangling the strategy adopted by these parasites for iron acquisition and utilization can be good targets for the development of antileishmanial drugs. Here, in this review, we will address how Leishmania parasites acquire and utilize iron and heme during infection to macrophages.

Blood meal analysis and molecular detection of mammalian Leishmania DNA in wild-caught Sergentomyia spp. from Tunisia and Saudi Arabia.

Phlebotomine sand flies (Diptera: Phlebotominae) belonging to the genus Phlebotomus are vectors of pathogens such as arboviruses, bacteria, and parasites (Leishmania). Species of the genus Sergentomyia (Se.) transmit Sauroleishmania (Reptile Leishmania) and feed on cold-blooded vertebrates; recently, they have been incriminated in mammalian Leishmania transmission. In addition, they have been reported to feed on warm-blooded vertebrates. This study aimed to (i) screen wild-caught Sergentomyia species for the detection of mammalian Leishmania and (ii) identify the blood meal origin of engorged females. The sand flies were collected using centers for disease control and prevention (CDC) traps, mounted and identified morphologically. Only females of the genus Sergentomyia were screened for Leishmania infection using PCR targeting the 18S ribosomal DNA locus. For positive specimens, Leishmania parasites were typed using nested PCR targeting ribosomal internal transcribed spacer 1 followed by digestion with HaeIII. The PCR-RFLP results were confirmed through sequencing. Blood meal identification was performed through PCR amplification of the vertebrate cytochrome b gene using degenerate primers followed by sequencing. In total, 6026 sand fly specimens were collected between 2009 and 2018. Among these, 511 belonged to five species of Sergentomyia genus: Se. minuta (58.51%), Se. fallax (18.01%), Se. clydei (14.68%), Se. dreyfussi (6.26%), and Se. antennata (2.54%). A total of 256 female Sergentomyia sp. specimens were screened for Leishmania infection. Seventeen (17) were positive (6.64%). Two Leishmania species were identified. Leishmania major DNA was detected in five specimens; this included three Se. fallax, one Se. minuta, and one Se. dreyfussi collected from Tunisia. Leishmania infantum/L. donovani complex was detected in four Se. minuta and three Se. dreyfussi specimens collected from Tunisia. In addition, we identified the blood meal origin of five engorged Se. minuta specimens collected from Tunisia. Sequencing results revealed two blood sources: humans (n = 4) and reptiles (n = 1) indicating possible role of Sergentomyia species in the transmission of human Leishmania. In addition, these species could be involved in the life cycle of L. infantum/L. donovani complex and L. major. The results of the blood meal origin showed that Sergentomyia fed on both cold- and warm-blooded vertebrates. These findings enable a better understanding of the behavior of this sand fly genus. Further studies should focus on the role of Sergentomyia in human Leishmania transmission and possible control of this disease.

Is leishmaniasis the new emerging zoonosis in the world?

Leishmania is a genus of parasitic protozoa that causes a disease called leishmaniasis. Leishmaniasis is transmitted to humans through the bites of infected female sandflies. There are several different species of Leishmania that can cause various forms of the disease, and the symptoms can range from mild to severe, depending on species of Leishmania involved and the immune response of the host. Leishmania parasites have a variety of reservoirs, including humans, domestic animals, horses, rodents, wild animals, birds, and reptiles. Leishmaniasis is endemic of 90 countries, mainly in South American, East and West Africa, Mediterranean region, Indian subcontinent, and Central Asia. In recent years, cases have been detected in other countries, and it is already an infection present throughout the world. The increase in temperatures due to climate change makes it possible for sandflies to appear in countries with traditionally colder regions, and the easy movement of people and animals today, facilitate the appearance of Leishmania species in new countries. These data mean that leishmaniasis will probably become an emerging zoonosis and a public health problem in the coming years, which we must consider controlling it from a One Health point of view. This review summarizes the prevalence of Leishmania spp. around the world and the current knowledge regarding the animals that could be reservoirs of the parasite.

Src- and Abl-family kinases activate spleen tyrosine kinase to maximize phagocytosis and Leishmania infection.

Leishmania spp. are obligate intracellular parasites that must be internalized by phagocytic cells to evade immune responses and cause disease. The uptake of both Leishmania promastigotes (insect-stage parasites) and amastigotes (proliferative-stage parasites in humans and mice) by phagocytes is thought to be mainly host cell driven, not parasite driven. Our previous work indicates that host Src- and Abl-family kinases facilitate Leishmania entry into macrophages and pathogenesis in murine cutaneous leishmaniasis. Here, we demonstrate that host spleen tyrosine kinase (SYK) is required for efficient uptake of Leishmania promastigotes and amastigotes. A Src-family kinase-Abl-family kinase-SYK signaling cascade induces Leishmania amastigote internalization. Finally, lesion size and parasite burden during Leishmania infection is significantly decreased in mice lacking SYK in monocytes or by treatment with the SYK inhibitor entospletinib. In summary, SYK is required for maximal Leishmania uptake by macrophages and disease in mice. Our results suggest potential for treating leishmaniasis using host cell-directed agents.

Antiproliferative Activity and Ultrastructural Changes in Promastigote and Amastigote forms of Leishmania amazonensis Caused by Limonene-Acylthiosemicarbazide Hybrids.

Leishmaniasis is a tropical zoonotic disease. It is found in 98 countries, with an estimated 1.3 million people being affected annually. During the life cycle, the Leishmania parasite alternates between promastigote and amastigote forms. The first line treatment for leishmaniasis are the pentavalent antimonials, such as N-methylglucamine antimoniate (Glucantime®) and sodium stibogluconate (Pentostam®). These drugs are commonly related to be associated with dangerous side effects such as cardiotoxicity, nephrotoxicity, hepatotoxicity, and pancreatitis. Considering these aspects, this work aimed to obtain a new series of limonene-acylthiosemicarbazides hybrids as an alternative for the treatment of leishmaniasis. For this, promastigotes, axenic amastigotes, and intracellular amastigotes of Leishmania amazonensis were used in the antiproliferative assay; J774-A1 macrophages for the cytotoxicity assay; and electron microscopy techniques were performed to analyze the morphology and ultrastructure of parasites. ATZ-S-04 compound showed the best result in both tests. Its IC50 , in promastigotes, axenic amastigotes and intracellular amastigotes was 0.35±0.08 µM, 0.49±0.06 µM, and 15.90±2.88 µM, respectively. Cytotoxicity assay determined a CC50 of 16.10±1.76 µM for the same compound. By electron microscopy, it was observed that ATZ-S-04 affected mainly the Golgi complex, in addition to morphological changes in promastigote forms of L. amazonensis.

Development of Leishmania Species Strains with Constitutive Expression of eGFP.

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people worldwide. Infection with L. donovani can result in fatal visceral disease. In Panama, Colombia, and Costa Rica, L. panamensis is responsible for most of the reported cases of cutaneous and mucocutaneous leishmaniasis. Studying a large number of drug candidates with the methodologies available to date is quite difficult, given that they are very laborious for evaluating the activity of compounds against intracellular forms of the parasite or for performing in vivo assays. In this work, we describe the generation of L. panamensis and L. donovani strains with constitutive expression of the gene that encodes for an enhanced green fluorescent protein (eGFP) integrated into the locus that encodes for 18S rRNA (ssu). The gene encoding eGFP was obtained from a commercial vector and amplified by polymerase chain reaction (PCR) to enrich it and add restriction sites for the BglII and KpnI enzymes. The eGFP amplicon was isolated by agarose gel purification, digested with the enzymes BglII and KpnI, and ligated into the Leishmania expression vector pLEXSY-sat2.1 previously digested with the same set of enzymes. The expression vector with the cloned gene was propagated in E. coli, purified, and the presence of the insert was verified by colony PCR. The purified plasmid was linearized and used to transfect L. donovani and L. panamensis parasites. The integration of the gene was verified by PCR. The expression of the eGFP gene was evaluated by flow cytometry. Fluorescent parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity were selected using flow cytometry.

Identification of infection by Leishmania spp. in wild and domestic animals in Brazil: a systematic review with meta-analysis (2001-2021).

Leishmaniasis is a zoonosis caused by protozoan species of the genus Leishmania. It generates different clinical manifestations in humans and animals, and it infects multiple hosts. Leishmania parasites are transmitted by sandfly vectors. The main objective of this systematic review was to identify the host, or reservoir animal species, of Leishmania spp., with the exception of domestic dogs, that were recorded in Brazil. This review included identification of diagnostic methods, and the species of protozoan circulating in the country. For this purpose, a literature search was conducted across index journals. This study covered the period from 2001 to 2021, and 124 studies were selected. Eleven orders possible hosts were identified, including 229 mammalian species. Perissodactyla had the highest number of infected individuals (30.69%, 925/3014), with the highest occurrence in horses. In Brazil, the most commonly infected species were found to be: horses, domestic cats, rodents, and marsupials. Bats, that were infected by one or more protozoan species, were identified as potential reservoirs of Leishmania spp. Molecular tests were the most commonly used diagnostic methods (94 studies). Many studies have detected Leishmania spp. (n = 1422): Leishmania (Leishmania) infantum (n = 705), Leishmania (Viannia) braziliensis (n = 319), and Leishmania (Leishmania) amazonensis (n = 141). Recognizing the species of animals involved in the epidemiology and biological cycle of the protozoan is important, as this allows for the identification of environmental biomarkers, knowledge of Leishmania species can improve the control zoonotic leishmaniasis.

Entomological Exploration of Sand Flies in Human Communities Affected by Cutaneous and Visceral Leishmaniasis in El Hajeb Province, Morocco.

Sand flies are the exclusive vectors of leishmaniasis. This group of parasitic diseases is a serious public health problem in Morocco. The aim of this study was to investigate the sand fly fauna, mainly the species composition, biodiversity, and seasonal activity of sand flies in El Hajeb in central Morocco. A total of six stations (Aït Naaman, Aït Rbaa, Aït Brahim, Ain Taoujdate, Sidi Mbarek, and Aït Oufella) were studied, five of which had recently recorded cases of leishmaniasis. Sand fly bimonthly captures were carried out using a sticky paper trap in different biotopes from March to December 2019. A total of 14590 adult sand flies were collected. The activity of the sand fly started in April and declined in November. The periods of high abundance were July, September, and November. Morphological identification of sand flies shows the presence of twelve species: Phlebotomus papatasi, P. longicuspis, P. perniciosus, P. sergenti, P. bergeroti, P. alexandri, P. dreyfussi, P. ariasi, Sergentomyia fallax, S. minuta, S. schwetzi, and S. antennata. The analysis showed that species belonging to the genus Phlebotomus were the most dominant (93.3%) and the risk periods were spread during the summer and autumn seasons. The present study provides for the first time information on the species of sand flies in El Hajeb. It, therefore, provides decision makers with an important tool to conduct vector control actions during peak periods in order to limit the transmission of leishmaniasis. A preprint was made available by the research square in the following link: "https://assets.researchsquare.com/files/rs-1409330/v1/dfef7013-0327-4a54-897f-214924a2d950.pdf?c=1646838874."

Optimization of Orally Bioavailable Antileishmanial 2,4,5-Trisubstituted Benzamides.

Leishmaniasis, a neglected tropical disease caused by Leishmania species parasites, annually affects over 1 million individuals worldwide. Treatment options for leishmaniasis are limited due to high cost, severe adverse effects, poor efficacy, difficulty of use, and emerging drug resistance to all approved therapies. We discovered 2,4,5-trisubstituted benzamides (4) that possess potent antileishmanial activity but poor aqueous solubility. Herein, we disclose our optimization of the physicochemical and metabolic properties of 2,4,5-trisubstituted benzamide that retains potency. Extensive structure-activity and structure-property relationship studies allowed selection of early leads with suitable potency, microsomal stability, and improved solubility for progression. Early lead 79 exhibited an 80% oral bioavailability and potently blocked proliferation of Leishmania in murine models. These benzamide early leads are suitable for development as orally available antileishmanial drugs.

Cysteine synthase: multiple structures of a key enzyme in cysteine synthesis and a potential drug target for Chagas disease and leishmaniasis.

Chagas disease is a neglected tropical disease (NTD) caused by Trypanosoma cruzi, whilst leishmaniasis, which is caused by over 20 species of Leishmania, represents a group of NTDs endemic to most countries in the tropical and subtropical belt of the planet. These diseases remain a significant health problem both in endemic countries and globally. These parasites and other trypanosomatids, including T. theileri, a bovine pathogen, rely on cysteine biosynthesis for the production of trypanothione, which is essential for parasite survival in hosts. The de novo pathway of cysteine biosynthesis requires the conversion of O-acetyl-L-serine into L-cysteine, which is catalysed by cysteine synthase (CS). These enzymes present potential for drug development against T. cruzi, Leishmania spp. and T. theileri. To enable these possibilities, biochemical and crystallographic studies of CS from T. cruzi (TcCS), L. infantum (LiCS) and T. theileri (TthCS) were conducted. Crystal structures of the three enzymes were determined at resolutions of 1.80 Šfor TcCS, 1.75 Šfor LiCS and 2.75 Šfor TthCS. These three homodimeric structures show the same overall fold and demonstrate that the active-site geometry is conserved, supporting a common reaction mechanism. Detailed structural analysis revealed reaction intermediates of the de novo pathway ranging from an apo structure of LiCS and holo structures of both TcCS and TthCS to the substrate-bound structure of TcCS. These structures will allow exploration of the active site for the design of novel inhibitors. Additionally, unexpected binding sites discovered at the dimer interface represent new potential for the development of protein-protein inhibitors.

Virus-Induced Lysis of Tumor and Other Pathogenic Unicellular Entities and Its Potential to Treat Leishmaniasis.

This article is focused on the main pathways used by viruses to achieve infection and lysis of unicellular eukaryotes described as pathogenic for multicellular organisms. In light of the recent discussions on how tumor cells exhibit unicellular behavior, highly malignant cells can be considered as another unicellular pathogenic entity, but with endogenous origin. Thus, a comparative panel of viral lysis of exogenous pathogenic unicellular eukaryotes such as Acanthamoeba sp., yeast, and tumors is presented. The important intracellular parasite Leishmania sp is also presented, which, in contrast, has its virulence improved by viral infections. The possible exploitation of viral-mediated eukaryotic cell lysis to overcome infections of Leishmania sp is discussed.

Development of a novel immunoFET technology-based POC assay for detection of Leishmania donovani and Leishmania major.

Leishmaniasis is considered as one of the 20 neglected tropical diseases. Current methods of leishmanial diagnosis depend on conventional laboratory-based techniques, which are time-consuming, costly and require special equipment and trained personnel. In this context, we aimed to provide an immuno field effect transistors (ImmunoFET) biosensor that matches the conventional standards for point-of-care (POC) monitoring and detection of Leishmania (L.) donovani/Leishmania major. Crude antigens prepared by repeated freeze thawing of L. donovani/L. major stationary phase promastigotes were used for ELISA and ImmunoFETs. Lesishmania-specific antigens were serially diluted in 1× PBS from a concentration of 106 -102 parasites/mL. A specific polyclonal antibody-based sandwich ELISA was established for the detection of Leishmania antigens. An immunoFET technology-based POC novel assay was constructed for the detection of Leishmania antigens. Interactions between antigen-antibody at the gate surface generate an electrical signal that can be measured by semiconductor field-effect principles. Sensitivity was considered and measured as the change in current divided by the initial current. The final L. donovani/L. major crude antigen protein concentrations were measured as 1.50 mg/mL. Sandwich ELISA against the Leishmania 40S ribosomal protein detected Leishmania antigens could detect as few as 100 L. donovani/L. major parasites. An immunoFET biosensor was constructed based on the optimization of aluminium gallium nitride/gallium nitride (AlGaN/GaN) surface oxidation methods. The device surface was composed by an AlGaN/GaN wafer with a 23 nm AlGaN barrier layer, a 2 µm GaN layer on the silicon carbide (SiC) substrate for Leishmania binding, and coated with a specific antibody against the Leishmania 40S ribosomal protein, which was successfully detected at concentrations from 106 to 102 parasites/mL in 1× PBS. At the concentration of 104 parasites, the immunoFETs device sensitivities were 13% and 0.052% in the sub-threshold regime and the saturation regime, respectively. Leishmania parasites were successfully detected by the ImmunoFET biosensor at a diluted concentration as low as 150 ng/mL. In this study, the developed ImmunoFET biosensor performed well. ImmunoFET biosensors can be used as an alternative diagnostic method to ELISA. Increasing the sensitivity and optimization of immuno-FET biosensors might allow earlier and faster detection of leishmaniasis.