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For the past 15 years, trials of combination therapy options for visceral leishmaniasis have been conducted with the aim of identifying effective, and safe treatment regimens that were shorter than existing monotherapy regimens and could also prevent or delay the emergence of drug resistance. Although first-line treatment currently relies on combination therapy in east Africa, this is not true in Latin America owing to disappointing trial results, with lower than expected efficacy seen for the combination treatment group. By contrast, several effective combination therapy regimens have been identified through trials on the Indian subcontinent; yet, first-line therapy is still AmBisome monotherapy as the drug is part of a free donation programme and is highly effective in this region. Achieving a short all-oral combination treatment will require new chemical entities, several of which are currently under evaluation. Future studies should systematically include pharmacological substudies to ensure optimal dosing for all patient groups. To achieve maximal impact of new combination treatments, mechanisms to ensure drug availability and access after trials should be established. Enhancing the longevity of current and novel treatments will require effective systems for early detection of emerging drug resistance. [ABSTRACT FROM AUTHOR]

Leishmania infections are global, occurring in 98 countries and all World Health Organization (WHO) regions with 600 million to 1 billion people at risk of infection. Visceral leishmaniasis is associated with almost 20,000 reported deaths annually, with children under 5 years of age being at the greatest risk of mortality. Amongst WHO-recognised Neglected Tropical Diseases (NTDs), leishmaniasis is one of the most important in terms of mortality and morbidity. With an increasing global burden of disease and a growing threat from climate change, urbanisation and drug resistance, there remains an imperative to develop leishmaniasis vaccines. New tools to understand correlates of protection and to assess vaccine efficacy are being developed to ease the transition into larger scale efficacy trials or provide alternate routes to licensure. Early indications suggest a diverse portfolio of manufacturers exists in endemic countries with an appetite to develop leishmaniasis vaccines. This Vaccine Value Profile (VVP) provides a high-level, comprehensive assessment of the currently available data to inform the potential public health, economic, and societal value of leishmaniasis vaccines. The leishmaniasis VVP was developed by a working group of subject matter experts from academia, public health groups, policy organizations, and non-profit organizations. All contributors have extensive expertise on various elements of the leishmaniasis VVP and have collectively described the state of knowledge and identified the current gaps. The VVP was developed using only existing and publicly available information. [ABSTRACT FROM AUTHOR]

• Essential genes (Pyridoxal kinase and sterol alpha-14 demethylase) are drug targets. • Discerned the atomistic interactions of small molecules with target proteins through Molecular Docking and MD Simulations. • Non-bonded contacts and MM-PBSA calculations aided in deciphering nitazoxanide and artemisinin dynamic interactions with the target proteins. • This biocomputing study identified plausible drug inhibitors against essential leishmanial genes. Leishmania donovani , an obligatory intracellular flagellate pathogen, is the underlying cause of visceral leishmaniasis (VL), a fatal disease that poses a significant challenge to existing therapeutic approaches and leads to human mortality. In an endeavor to find an antileishmanial drug to combat VL, we aimed to assess the approved drug molecules against the specific drug targets of VL. In this study, a theoretical method was used to select two essential therapeutic targets (pyridoxal kinase [PK] and sterol alpha-14 demethylase [SDM]) which were present in both the data set of essential genes and drug target proteins. The selected PK and SDM proteins in L. donovani play pivotal roles as essential enzymes in the crucial vitamin B6 salvage and sterol biosynthesis pathways, respectively, leading to pathogenicity in humans. In addition to that drugs were gathered from the DrugBank and Drug Central databases and 325 (out of 4867) compounds having anti-parasitic properties were screened by PASS analysis. Consequently, three ligands (referred to as Lig_1, Lig_2, and Lig_3) were chosen based on their elevated Pa values, docking scores, and notable medicinal applications. Moreover, the result obtained from MD simulation suggests Lig_1 [Nitazoxanide (PubChem ID-41684)] does not affect the structural integrity of both targets. Additionally, evaluation of total binding energies by MMPBSA analysis showed stronger binding of Lig_1 with PK and SDM is -100.71 and -175.61 kJ/mol, respectively compared to others. As a whole, the methodology employed in this research involves the simultaneous identification of suitable protein targets and potential inhibitors. Through this investigation, we have demonstrated that compounds derived from a biocomputing approach exhibit interaction mechanisms as inhibitors against drug targets, offering a promising avenue for addressing VL. [ABSTRACT FROM AUTHOR]

Post kala-azar dermal leishmaniasis (PKDL) is the only dermal form of leishmaniasis that presents with hypopigmentation comparable with vitiligo, leprosy, and pitryasis versicolor. In the absence of an animal model, the mechanism(s) responsible for melanocyte loss/dysfunction in PKDL is/are poorly defined. Although the destruction of melanocytes is considered multifactorial, immune dysregulation is one of the key contributory players in this and other hypopigmentary dermatoses. Understanding of the complex interplay between immune cells and mediators, leading to the loss/destruction of melanocytes in PKDL, could translate into 'host-directed' chemotherapeutic strategies to prevent the stigma associated with hypopigmentation. Post kala-azar dermal leishmaniasis (PKDL), a heterogeneous dermal sequela of visceral leishmaniasis (VL), is challenging in terms of its etiopathogenesis. Hypopigmentation is a consistent clinical feature in PKDL, but mechanisms contributing to the loss of melanocytes remains poorly defined. Like other hypopigmentary dermatoses – for example, vitiligo, psoriasis, and leprosy – the destruction of melanocytes is likely a multifactorial phenomenon, key players being immune dysregulation and inflammation. This review focuses on immunological mechanisms responsible for the 'murder' of melanocytes, prime suspects at the lesional sites being CD8+ T cells and keratinocytes and their criminal tools being proinflammatory cytokines, for example, IFN-γ, IL-6, and TNF-α. Collectively, these may cause decreased secretion of melanocyte growth factors, loss/attenuation of cell adhesion molecules and inflammasome activation, culminating in melanocyte death. [ABSTRACT FROM AUTHOR]

The drug discovery pipeline for leishmaniasis and trypanosomiasis has been filling with novel chemical entities with known mechanisms of action. González et al. and Braillard et al. report a cytochrome bc 1 complex inhibitor as another promising preclinical candidate for visceral leishmaniasis (VL) and, in combination with benznidazole, for chronic Chagas' disease (CCD). [ABSTRACT FROM AUTHOR]

• Severe visceral leishmaniasis with non-ulcerated cutaneous leishmaniasis. • A Crithidia -related parasite was isolated from bone marrow aspirate and spleen. • Whole-genome sequencing of clinical isolates suggested a novel parasite species. • Clinical implications of this trypanosomatid co-infection remain unknown in visceral leishmaniasis. [Display omitted] We report a refractory and relapsed visceral leishmaniasis case in a male child patient followed from 2016 to 2020, whose clinical isolates from multiple relapses were analyzed at the genome level. To the best of our knowledge, it is the first report that both visceral leishmaniasis and non-ulcerated cutaneous leishmaniasis have concomitantly manifested in the same patient. Importantly, sequence analysis revealed that the patient was co-infected with Leishmania infantum and a Crithidia -related parasite, which was previously found in a fatal case of visceral leishmaniasis from the same endemic region. [ABSTRACT FROM AUTHOR]

Organ transplantation can lead to human visceral leishmaniasis (VL) transmission in humans. This report aims to describe the possible complications related to an atypical course of VL after kidney transplantation. A 61-year-old man who suffered end-stage renal failure received a deceased donor kidney transplant after 2 years of hemodialysis. Tacrolimus, mycophenolate mofetil, and prednisolone were used for immunosuppressive therapy, and renal function remained stable for 2.5 years. He was referred to our hospital because of fever and malaise. Physical and radiological examinations showed mild splenomegaly and cervical and inguinal lymphadenopathy. Laboratory data showed bicytopenia, elevated C-reactive protein, serum creatinine, and non-nephrotic proteinuria. Bone marrow biopsy aspiration showed no abnormality. Polymerase chain reaction confirmed the diagnosis of Leishmania infantum. Anti-leishmanial therapy was initiated with liposomal amphotericin B for 2 weeks, and the patient became clinically stable. So far, there has been no evidence of clinical or biological relapse, and kidney function is stable. Considering that VL has become increasingly widespread in immunocompromised patients in endemic regions, especially in patients with transplants, it is crucial to screen and rule out VL as a cause of infection in these patients. The probability of this problem should be considered in every patient with a transplant in endemic and nonendemic areas. Furthermore, our study showed that through timely diagnosis using noninvasive methods and standard treatments, mortality caused by this disease can be properly prevented. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a tropical parasitic disease caused by Leishmania spp. They cause several presentations of illness ranging from cutaneous leishmaniasis to visceral leishmaniasis. The current arsenal of drugs to treat leishmaniasis is limited, and drug resistance further impedes the problem. Therefore, it is necessary to revisit the available information to identify an alternative or new target for treatment. The glycoprotein 63 (gp63), is a potential anti-leishmanial target that plays a significant role in host-pathogen interaction and virulence. Many studies are ongoing to develop gp63 inhibitors or use it as a vaccine target. In this review, we will discuss the potential of gp63 as a drug target. This review summarises the studies focusing on gp63 as a drug target and its inhibitors identified using in silico approaches. [Display omitted] • gp63 a potential drug and vaccine target. • Active site is conserved in all Leishmania spp. • May serve as pan drug target. • In silico studies identify its inhibitors. [ABSTRACT FROM AUTHOR]

Vaccination against visceral leishmaniasis (VL) should be considered as a safe and effective measure to disease control; however, few vaccines are available against canine VL and there is no an approved human vaccine. In this context, in the present study, we evaluated the endonuclease III (ENDO) protein, which was recently showed to be antigenic for human disease, as a vaccine candidate against Leishmania infantum infection. The recombinant protein (rENDO) was administered in BALB/c mice alone or associated with saponin (rENDO/Sap) or micelles (rENDO/Mic) as adjuvants. Controls received saline, saponin or empty micelles. Results showed that both rENDO/Sap and rENDO/Mic compositions induced higher levels of IFN-γ, IL-12, TNF-α, and GM-CSF cytokines, besides nitrite and IgG2a isotype antibodies, before and after challenge infection, which were related to both CD4+ and CD8+ T cell subtypes. The immunological results contributed to significant reductions in the parasite load found in the spleens, livers, bone marrows and draining lymph nodes of the vaccinated animals. In general, mice immunized with rENDO/Mic presented a slightly higher Th1-type cellular and humoral immune response, as compared to those receiving rENDO/Sap. In addition, saponin caused a slight to moderate inflammatory edema in their vaccinated footpads, which was not observed when micelles were used with rENDO. In addition, a preliminary analysis showed that the recombinant protein was immunogenic to human cells cultures, since PBMCs from treated VL patients and healthy subjects showed higher lymphoproliferation and IFN-γ production in the culture supernatants. In conclusion, data suggest that rENDO could be considered as a candidate to be evaluated in future studies as vaccine to protect against VL. [Display omitted] • Endonuclease III (ENDO) was evaluated as vaccine candidate against L. infantum. • Saponin and polymeric micelles were used as Th1-type adjuvants. • Mice immunized with ENDO plus adjuvants mounted a specific Th1-type response. • Parasitological protection was reached using both vaccine compositions. • Endonuclease III induced in vitro immunogenicity in human cells. [ABSTRACT FROM AUTHOR]

A clear understanding of the accuracy of diagnostic tests is essential for the management of infections such as visceral leishmaniasis in endemic areas. Using both published and unpublished datasets from field diagnostic trials of visceral leishmaniasis in the past ten years, we show the potential effects of unrecognised sources of bias including work-up bias, referral bias, and reference test bias on the results. We outline why these biases, which can occur in diagnostic studies of any disease, can go unrecognised despite adherence to current STARD and QUADAS-2 guidelines. Using these examples and referring to others seen in studies of bacterial and viral infections, we make specific recommendations on how these biases might be avoided through specific steps in study design, study reporting, and the locations where studies are conducted. [ABSTRACT FROM AUTHOR]

Iron (Fe) sequestration is one of the most important strategies of the host to control the growth and survival of invading pathogens. Ferritin (Ft) plays a pivotal role in the sequestration mechanism of mammalian hosts by storing Fe. Recent evidence suggests that poly(rC)-binding proteins (PCBPs) act as chaperones for loading Fe into Ft. Incidentally, modulation of host PCBPs in respect to storing Fe in Ft during any infection remains unexplored. Among PCBPs, PCBP1 and PCBP2 are present in every cell type and involved in interacting with Ft for Fe loading. Leishmania donovani (LD) resides within macrophages during the mammalian stage of infection, causing life-threatening visceral leishmaniasis. Here, we reveal the ability of LD to cleave PCBP1 and PCBP2 in host monocytes/macrophages. LD cleaves PCBP1-FLAG into two fragments and PCBP2-FLAG into multiple fragments, thus affecting their in-teractions with Ft and resulting in decreased Fe loading into Ft. LD-derived culture supernatant or exosome-enriched fractions are also able to cleave PCBPs, suggesting involvement of a secreted protease of the parasite. Using an immune-depletion experiment and transgenic mutants, we confirmed the involvement of zinc-metalloprotease GP63 in cleaving PCBPs. We further revealed that by cleaving host PCBPs, Leishmania could inhibit Fe loading into Ft to accumulate available Fe for higher intracellular growth. This is the first report of a novel strategy adopted by a mammalian pathogen to interfere with Fe sequestration into Ft by cleaving chaperones for its survival advantage within the host. [ABSTRACT FROM AUTHOR]

Dogs are the most common domestic reservoir of Leishmania infantum , making canine visceral leishmaniasis (CVL) a serious public health issue. Identifying new methodologies that can mimic lymphoid and myeloid competence in naturally infected dogs could lower costs and save time in preliminary screenings of potential immunotherapeutic agents and vaccines against CVL. For that, we established a cell-to-cell communication approach between lymphocytes and myeloid cells from healthy, asymptomatic (infected, without apparent clinical signs) and symptomatic (infected with apparent clinical signs) dogs. Peripheral blood mononuclear cells (PBMC) from these dogs were used as source of CD4+, CD8+ T lymphocytes and macrophages, that were posteriorly infected with L. infantum GFP+ promastigotes (green fluorescent protein). Macrophages co-cultured with purified lymphocytes were tested for the ability to control cellular parasitism, and their microbicidal function by producing nitric oxide (NO) and reactive oxygen species (ROS). The kind of T cell response within the co-culture was also evaluated, by assessing their ability to produce interferon-gamma (IFN-γ) and interleukin 4 (IL-4). The data suggests that T lymphocytes from symptomatic dogs are more prone to produce IL-4 than the ones from asymptomatic dogs. Macrophages from asymptomatic dogs also demonstrated a higher microbicidal potential, with increased levels of NO and ROS production, compared to symptomatic dogs, mainly in highly parasitized cells. Together, our results identify the ratio of IL-4/IFN-γ produced by CD4+ and CD8+ T cells, as well as, the ratio between parasite GFP signal/NO and ROS signal in macrophages as potential immunological biomarkers of failure and success of the screened agents. Our findings also propose a reliable methodology that can be used to follow the immune response in trials of potential drugs or vaccines targeting CVL. [Display omitted] • Leishmania infantum -infected macrophages control parasites more efficiently when co-cultured with CD4+ and CD8+ T cells. • Heterologous co-culture from asymptomatic dogs has lower parasite burden than cultures from symptomatic dogs. • Co-culture system from dogs with CVL is a promising tool for screening immunotherapeutic agents for such disease. [ABSTRACT FROM AUTHOR]

[Display omitted] • Leishmania infantum is the only Leishmania species circulating in Italy. • In the Emilia-Romagna region, humans and dogs are infected by two distinct L. infantum strains. • For the first known time L. infantum infection was recorded in ruminants (roe deer). • Over 90% of L. infantum infections in deer are associated with the "human" strain. • This result suggests that these animals are a novel reservoir for human visceral leishmaniasis. Leishmaniasis is a complex human disease caused by intracellular parasites of the genus Leishmania, predominantly transmitted by the bite of sand flies. In Italy, leishmaniasis is caused exclusively by Leishmania infantum , responsible for the human and canine visceral leishmaniases (HVL and CVL, respectively). Within the Emilia-Romagna region, two different foci are active in the municipalities of Pianoro and Valsamoggia (both in the province of Bologna). Recent molecular studies indicated that L. infantum strains circulating in dogs and humans are different, suggesting that there is an animal reservoir other than dogs for human visceral leishmaniasis in the Emilia-Romagna region. In this work, we analyzed specimens from wild animals collected during hunts or surveillance of regional parks near active foci of human visceral leishmaniasis for L. infantum infection in the province of Bologna. Out of 70 individuals analyzed, 17 (24%) were positive for L. infantum. The infection prevalence in hedgehogs (Erinaceus europaeus), roe deer (Capreolus capreolus), badgers (Meles meles), and bank voles (Myodes glareolus) was 80, 33, 25, and 11%, respectively. To distinguish the two strains of L. infantum we have developed a nested PCR protocol optimized for animal tissues. Our results demonstrated that most (over 90%) of L. infantum infections in roe deer were due to the strain circulating in humans in the Emilia-Romagna region. [ABSTRACT FROM AUTHOR]

In recent years, several advances have been observed in vaccinology especially for neglected tropical diseases (NTDs). One of the tools employed is epitope prediction by immunoinformatic approaches that reduce the time and cost to develop a vaccine. In this scenario, immunoinformatics is being more often used to develop vaccines for NTDs, in particular visceral leishmaniasis (VL) which is proven not to have an effective vaccine yet. Based on that, in a previous study, two predicted T-cell multi-epitope chimera vaccines were experimentally validated in BALB/c mice to evaluate the immunogenicity, central and effector memory and protection against VL. Considering the results obtained in the mouse model, we assessed the immune response of these chimeras in Mesocricetus auratus hamster, which displays, experimentally, similar pathological status to human and dog VL disease. Our findings indicate that both chimeras lead to a dominant Th1 response profile, inducing a strong cellular response by increasing the production of IFN-γ and TNF-α cytokines associated with a decrease in IL-10. Also, the chimeras reduced the spleen parasite load and the weight a correlation between protector immunological mechanisms and consistent reduction of the parasitic load was observed. Our results demonstrate that both chimeras were immunogenic and corroborate with findings in the mouse model. Therefore, we reinforce the use of the hamster as a pre-clinical model in vaccination trials for canine and human VL and the importance of immunoinformatic to identify epitopes to design vaccines for this important neglected disease. [ABSTRACT FROM AUTHOR]

• Leishmania infantum is the etiologic agent of zoonotic visceral leishmaniasis, which can be fatal. • Visceral leishmaniasis treatment may be toxic, and resistance has been reported. • There are currently no vaccines available for humans. • Transgenic L. infantum could be an excellent vaccine candidate. • Prime-boosted mice have shown maximized protection. The etiologic agents of visceral leishmaniasis are Leishmania infantum and Leishmania donovani. Despite the variety of drugs available to treat leishmaniasis, most lead to serious adverse effects, and resistance to these drugs has been reported. Currently, no leishmaniasis vaccine is available for humans. That is why the group developed transgenic L. infantum promastigote lines, which express toxic proteins after differentiation into amastigotes. That is why group developed the pFL-AMA plasmid and transfected it into L. Infantum promastigotes. This plasmid was expressed only in the amastigote form of the parasite. Sequences encoding toxic proteins (active bovine trypsin and egg avidin) were inserted in this plasmid, and the transfected parasites died after the differentiation process. In this study, two immunization protocols were performed in BALB/c mice: prime and prime-boost immunization prior to challenge with the wild-type L. infantum (WT). The parasite burdens in the spleen, liver, and bone marrow were evaluated to verify immunological protection. Histopathological analysis of the spleen and liver and the humoral immune response were also performed. The data showed that the parasite burden was reduced in prime-boosted mice in the spleen, liver, and bone marrow, indicating that mice immunized with two doses of the transfected parasites were satisfactorily protected. High levels of IgG, IgG1, and IgG2a antibodies were observed, as well as the presence of anti-inflammatory cytokine Interleukine-10 and pro-inflammatory cytokine Tumor Necrosis Factor-α (TNF-α) and Interferon-γ (IFN – γ) suggesting a Th1/Th2 mix response, in addition to the presence of multinucleated giant cells in the spleen and lymphocyte infiltration in the liver. Therefore, L. infantum transfected with a toxic plasmid is an excellent vaccine candidate against visceral leishmaniasis and the application of a boost before the challenge promotes greater protection against WT L. infantum infection. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is a fatal form among all forms of leishmaniasis and is caused by visceralization of the Leishmania donovani (Ld) parasite to the critical organs. Mild to severe malnutrition is common in VL patients and the deficiency of retinoic acid (RA), an important micronutrient, results in a compromised state of immune response in macrophages (mφ) leading to the increased parasite load. In the continuation of our earlier work, we observed loss of cellular cholesterol in infected mφ in the absence of RA i.e., upon inhibition of RALDH pathway. Moreover, the Leishmania utilizes host cholesterol for the establishment of infection and causes a decrease in the expressions of Niemann-Pick C2 (npc2) and Niemann-Pick C1 (npc1) genes involved in the uptake of extracellular cholesterol. This results in reduced levels of cellular cholesterol in infected mφ. Intrigued by this, as the first sign of our hypothesis, we investigated the presence of RA Response Element (RARE) sequences in the upstream of npc1 and npc2 genes. To functionally confirm this, we measured their expressions and the levels of cellular cholesterol in Ld infected mφ in the absence (i.e., using an inhibitor of RALDH pathway) and presence of RA. We found restoration of the levels of cellular cholesterol in infected mφ under the supplementation of RA resulting in the decreased parasite load. Hence, the supplementation of RA with the standard therapy and/or preventive use of RA could be potentially an advancement in the treatment and cure of VL patients. • Leishmania donovani infection impairs uptake of cholesterol. • Retinoic acid (RA) increases the expressions of genes involved in cholesterol uptake. • RA restores the levels of cellular cholesterol in Leishmania donovani infected mφ. • Treatment with RA reduces the parasite burden in infected mφ. [ABSTRACT FROM AUTHOR]

• Visceral leishmaniasis is a serious complication, especially in immunosuppressed patients, and is uncommon after liver transplantation. • The case report highlights the challenge of early diagnosis of visceral leishmaniasis in liver transplant recipients with atypical manifestations. • The aim of the report is to emphasize the suspicion of visceral leishmaniasis in symptomatic patients from emerging countries and to question the benefit of including protozoan screening in liver transplant donors and recipients in endemic areas. Leishmaniasis is a disease caused by a protozoan and transmitted by sandfly species in several emerging countries. Visceral leishmaniasis is a serious complication, especially in immunosuppressed patients, and is uncommon after liver transplantation. We report the case of a 48-year-old female patient who underwent liver transplantation owing to polycystic liver disease. Six months after the procedure, she was hospitalized with diarrhea, acute kidney failure, and leukopenia. She had been off steroids for 3 months and was taking mycophenolate and tacrolimus. She had already been treated for cytomegalovirus, which was negative on admission. During hospitalization, fever, splenomegaly, ascites, and pancytopenia appeared. Serology for Leishmania by indirect immunofluorescence was negative. Then, bone biopsy and molecular testing for Leishmania diagnosed it as visceral leishmaniasis. Amphotericin therapy was initiated with resolution of fever after 4 days of treatment and gradual recovery from pancytopenia. This case highlights the challenge of early diagnosis of visceral leishmaniasis in liver transplant recipients with diarrhea and leukopenia, which can be caused by immunosuppression or more prevalent viral diseases. Late onset of fever, splenomegaly, and a first negative serologic test also made early diagnosis difficult. The aim of the report is to emphasize the suspicion of visceral leishmaniasis in symptomatic patients from emerging countries and to question the benefit of including protozoan screening in liver transplant donors and recipients in endemic areas. [ABSTRACT FROM AUTHOR]

• Flame synthesis and characterisation of CoFe 2 O 4 -C60NP. • Synthesis and characterisation of A2 peptide antigen using LCMS and HPLC. • A2 peptide antigen decorated CoFe 2 O 4 -C60NP immunoelectrode. • Immunosensor with 30.34 fg/mL LOD at a linear range of 10−10 to 10−1 µg/mL. • Label-free electrochemical detection of A2-antibodies in spiked blood serum. Diagnosis of Visceral Leishmaniasis is challenging due to the shared clinical features with malaria, typhoid, and tuberculosis. A CoFe 2 O 4 -C60 nanocomposite-based immunosensor decorated with a sensitive A2 peptide antigen was fabricated to detect anti-A2 antibodies for application in visceral leishmaniasis diagnosis. The flame-synthesised nanocomposite was characterised using Fourier Transform Infrared spectroscopy (FTIR), X-ray diffraction spectroscopy (XRD), Scanning electron microscopy (SEM), Energy dispersive X-ray spectroscopy (EDX), Raman spectroscopy and electrochemical impedance spectroscopy (EIS) techniques. N terminated specific A2 peptide epitope antigen (NH 2 -QSVGPLSVGP-OH) was synthesised and characterised by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS). Using EDC/NHS, A2 peptide antigen (Apg) was immobilised on the CoFe 2 O 4 -C60-modified electrode. The performance of the immunosensor, Apg-CoFe 2 O 4 -C60NP/GCE, was evaluated by testing its ability to detect varying concentrations of anti-A2 antibody solution in PBS and spiked serum with 1 mM [Fe(CN) 6 ]3−/4− in 0.01 M PBS (pH 7.4) as supporting electrolyte. using differential pulse voltammetry. The immunosensor showed excellent reproducibility and a linear range of 10−10–10−1 µg/mL, with an experimental detection limit of 30.34 fg/mL. These results suggest that the fabricated sensor has great potential as a tool for diagnosing visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

In visceral and mucocutaneous leishmaniasis, humoral immune response can reflect disease severity and parasite burden. Cutaneous leishmaniasis (CL) in Sri Lanka is caused by a usually visceralizing parasite, Leishmania donovani. We assessed the parasite burden (relative quantity—RQ) in 190 CL patients using quantitative real-time PCR (qPCR—with primers designed for this study) and smear microscopy, then correlated it with clinical parameters and IgG response. RQ of parasite DNA was determined with human-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control. The qPCR sensitivity was tested with serially diluted DNA from cultured L. donovani parasites. Smears were assigned a score based on number of parasites per high power field. Data from previous studies were used for comparison and correlation; nested Internal Transcribed Spacer 1 (ITS1) PCR as reference standard (RS) and IgG antibody titers to the Leishmania rKRp42 antigen as the immune response. The qPCR amplified and quantified 86.8% of the samples while demonstrating a fair and significant agreement with ITS1-PCR and microscopy. Parasite burden by qPCR and microscopy were highly correlated (r = 0.76; p = 0.01) but showed no correlation with the IgG response (r = 0.056; p = 0.48). Corresponding mean RQs of IgG titers grouped by percentiles, showed no significant difference (p = 0.93). Mean RQ was higher in early lesions (p = 0.04), decreased with lesion size (p = 0.12) and slightly higher among papules, nodules and wet ulcers (p = 0.72). Our study established qPCR's efficacy in quantifying parasite burden in Sri Lankan CL lesions but no significant correlation was observed between the parasite burden and host IgG response to the Leishmania rKRP42 antigen. [Display omitted] • qPCR effectively quantified parasite burden in Sri Lankan cutaneous leishmaniasis. • Microscopic- and qPCR-based quantification of parasite burden correlated well. • Parasite burden and anti-rKRP42 IgG level showed no correlation in cutaneous form. • Parasite burden was higher in early lesions, decreasing with lesion size. [ABSTRACT FROM AUTHOR]

Leishmania , a protozoan parasite, is responsible for the occurrence of leishmaniasis, a disease that is prevalent in tropical regions. Visceral Leishmaniasis (VL), also known as kala-azar in Asian countries, is one of the most significant forms of VL, along with Cutaneous Leishmaniasis (CL) and Mucocutaneous Leishmaniasis (ML). Management of this condition typically entails the use of chemotherapy as the sole therapeutic option. The current treatments for leishmaniasis present several drawbacks, including a multitude of side effects, prolonged treatment duration, disparate efficacy across different regions, and the emergence of resistance. To address this urgent need, it is imperative to identify alternative treatments that are both safer and more effective. The identification of appropriate pharmacological targets in conjunction with biological pathways constitutes the initial stage of drug discovery. In this review, we have addressed the key metabolic pathways that represent potential pharmacological targets as well as prominent treatment options for leishmaniasis. • Target-based drug discovery. • Essential metabolic pathways in Leishmania like sterol biosynthesis, trypanothione route, glycolytic, Protein kinases, arginase and polyamine biosynthesis, trypanothione synthase and trypanothione reductase, topoisomerases, purine salvage, mannose biosynthesis etc. that are possible pharmacological targets. • Some important drugs for the treatment of leishmaniasis. [ABSTRACT FROM AUTHOR]

The present work aimed to develop and evaluate AmB-loaded nano-emulsion (AmB-NE) which will augment the solubility of AmB and lead to enhanced anti-leishmanial activity. The composition of AmB-NE was optimized by systematic screening followed by DoE-extreme vertices mixture design. The optimized NE revealed mean droplet size and PDI of 44.19 ± 5.5 nm, 0.265 ± 0.0723, respectively. The NE could efficiently encapsulate AmB with drug content and efficiency 83.509 ± 0.369% and 81.659 ± 0.013%, respectively. The presence of cholesterol and stearyl amine retarded the release (P < 0.0001) of AmB significantly compared to AmB suspension. The AmB-NE and pure AmB suspension demonstrated the IC 50 of 0.06309 μg/mL and 0.3309 μg/mL against L. donovani promastigotes after 48 h incubation. The formulation was robust at all exaggerated stability conditions such as freeze-thaw and centrifugation. These findings indicate that AmB-NE is an attractive approach to treat visceral leishmaniasis with improved activity. [Display omitted] • Amphotericin-loaded nanoemulsion was developed via extreme vertices mixture design. • Average size and polydispersity index was 44.19 ± 5.5 nm, 0.265 ± 0.072, respectively. • Inclusion of cholesterol and stearylamine imparted sustained release character. • Nanoemulsion caused significantly higher activity against L. donovani promastigotes. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis is a potentially fatal disease caused by infection by the intracellular protist pathogens Leishmania donovani or Leishmania infantum. Present therapies are ineffective because of high costs, variable efficacy against different species, the requirement for hospitalization, toxicity and drug resistance. Detailed analysis of previously published hit molecules suggested a crucial role of 'guanidine' linkage for their efficacy against L. donovani. Here we report the design of 2-aminoquinazoline heterocycle as a basic pharmacophore-bearing guanidine linkage. The introduction of various groups and functionality at different positions of the quinazoline scaffold results in enhanced antiparasitic potency with modest host cell cytotoxicity using a physiologically relevant THP-1 transformed macrophage infection model. In terms of the ADME profile, the C7 position of quinazoline was identified as a guiding tool for designing better molecules. The good ADME profile of the compounds suggests that they merit further consideration as lead compounds for treating visceral leishmaniasis. [Display omitted] • HTS, design, optimization gave substituted 2-aminoquinazolines as L. donovani inhibitors. • Scaffold-hopping gave leads with improved potency against L. donovani Vs hit 1. • Lead compounds exhibited better potency compared to Miltefosine. • Lead compounds displayed a good selectivity index against THP-1 macrophages. • Microwave-assisted ArSN reaction at C2 position of quinazoline derivatives. [ABSTRACT FROM AUTHOR]

• Growth of Leishmania parasites is inhibited by clotrimazole. • Clotrimazole effect was reversed by ergosterol supplementation. • Clotrimazole modulates cell membrane potential and its permeability. • Generates mitochondrial superoxide and causes mitochondrial depolarization. • Clotrimazole treated cells are arrested at sub G 0 stage of cell cycle. Clotrimazole is an FDA approved drug and is widely used as an antifungal agent. An extensive body of research is available about its mechanism of action on various cell types but its mode of killing of Leishmania donovani parasites is unknown. L. donovani causes Visceral Leishmaniasis which is a public health problem with limited treatment options. Its present chemotherapy is expensive, has adverse effects and is plagued with drug resistance issues. In this study we have explored the possibility of repurposing clotrimazole as an antileishmanial drug. We have assessed its efficacy on the parasites and attempted to understand its mode of action. We found that it has a half-maximal inhibitory concentration (IC 50) of 35.75 ± 1.06 μM, 12.75 ± 0.35 μM and 73 ± 1.41 μM in promastigotes, intracellular amastigotes and macrophages, respectively. Clotrimazole is 5.73 times more selective for the intracellular amastigotes as compared to the mammalian cell. Effect of clotrimazole was reduced by ergosterol supplementation. It leads to impaired parasite morphology. It alters plasma membrane permeability and disrupts plasma membrane potential. Mitochondrial function is compromised as is evident from increased ROS generation, depolarized mitochondrial membrane and decreased ATP levels. Cell cycle analysis of clotrimazole treated parasites shows arrest at sub-G 0 phase suggesting apoptotic mode of cell death. [Display omitted] [ABSTRACT FROM AUTHOR]

• There is a positive association between VL and El Niño, when compared to La Niña. • VL incidence was significant in Brazil's central and Northeast and Colombia's Northeast regions. • There is an impressive% of area suitable for the vector Lu. longipalpis in the Amazon region. • The potential distribution of Lu. longipalpis expanded especially in Roraima, Pará, Acre, and Mato Grosso. Visceral Leishmaniasis (VL) is the most severe of the three forms of Leishmaniasis. In the Americas, Brazil and Colombia present more than 90 % of the cases in the region. Our aim in this research was to estimate the association of the incidence rate of Visceral Leishmaniasis with the following environmental variables: the percentage of area suitable for the vector Lutzomyia longipalpis, t he episodes of La Niña and El Niño, the Brazilian and Colombian biomes. Epidemiological data were obtained from the Brazilian Notifiable Diseases Information System and the Colombian National Public Health Surveillance System. Environmental data were downloaded from the NASA Giovanni web app, the Modis Sensor database, and the meteorological agencies of Australia, Japan, and the United States of America. Records of the presence of Lu. longipalpis were obtained from public databases and previous studies. As a result, the incidence per 10,000 inhabitants with LEBS for each El Niño-Southern Oscillation (ENSO) episode showed the largest values during El Niño 2015–2016, mainly in Brazil's Northeast and Central regions and the Northeast region of Colombia. Compared with the Neutral 2012–2014 episode, the episodes of El Niño 2015–2016 and La Niña 2010–2011 showed an average increase in the monthly incidence rate of VL, and the average increase was higher during El Niño 2015–2016 (aIRR = 2.304 vs.1.453) We found a positive association between the incidence rate of VL and the El Niño 2015–2016 episode and an impressive% of area suitable for the vector Lu. longipalpis in the Amazon region.An increase of 1 % in the area suitable for the vector Lu. longipalpis leads to an average rise of 0.8 % in the monthly incidence rate of VL. Our study shows a possible association between VL incidence and ENSO, with the most considerable incidence rates observed during El Niño 2015–2016 in Brazil's Northeast and Central regions and the Northeast region of Colombia. The present study is very important to better understand the Visceral Leishmaniasis transmission dynamics. [ABSTRACT FROM AUTHOR]

• The age of mice directly affects leishmania parasitemia. • Use of the LaAg vaccine intranasally helps in the process of controlling parasite in young and elderly mice. • The process of controlling L. infantum infection is directly related to the expression of inhibitory receptors such as PD-1 and KLRG-1. There are no approved vaccines yet for human visceral leishmaniasis (VL), the most severe form of the leishmaniasis clinical manifestations that is fatal in over 95 % of untreated cases. It is well-accepted that immunological changes during aging have deleterious impact on the efficacy of vaccines and response to infections. In this work, we compared the response of young and aged mice to intranasal vaccination with killed Leishmania amazonensis promastigote antigens (LaAg) that were then challenged with L. infantum infection, a species that causes visceral leishmaniasis. Intranasal vaccination with LaAg induced a similar reduction in parasitism and hepatosplenomegaly in both young and aged mice compared to their unvaccinated counterparts. Following infection, there was also a less prominent inflammatory profile particularly in the vaccinated aged group, with lower production of TNF-α and nitrite compared to the respective unvaccinated group. Interestingly, the LaAg intranasal vaccination promoted increased production of IFN-γ that was observed in both young- and aged vaccinated groups. Additionally, CD4+ and CD8+ T cells from both vaccinated groups presented decreased expression of the inhibitory receptors PD-1 and KLRG1 compared to their unvaccinated controls. Interestingly, a strong positive correlation was observed between the expression of both inhibitory receptors PD-1 and KLRG1 and parasitism, which was more conspicuous in the unvaccinated-aged mice than in the others. Overall, this study helps define new strategies to improve vaccine effectiveness and provides a perspective for prophylactic alternatives against leishmaniasis. [Display omitted] [ABSTRACT FROM AUTHOR]

Herein, carboxymethyl chitosan (CMC) grafted lipid nanoformulations were facilely prepared by thin-film hydration method as a highly efficient biocompatible anti-leishmanial carrier encapsulating amphotericin B (AmB). Nanoformulations were characterized for their physicochemical characteristics wherein TEM analysis confirmed the spherical structure, whereas FTIR analysis revealed the conjugation of CMC onto nanoformulations and confirmed the free state of AmB. Furthermore, the wettability study confirmed the presence of CMC on the surface of nanoformulations attributed to the enhanced hydrophilicity. Surface hydrophilicity additionally contributes towards consistent mucin retention ability for up to 6 h, superior mucoadhesiveness, and hence enhanced bioavailability. The proposed nanoformulations with high encapsulation and drug loading properties displayed controlled drug release in the physiological microenvironment. In vitro, antileishmanial results showed an astounding 97% inhibition in amastigote growth. Additionally, in vivo studies showed that treatment with nanoformulations significantly reduced the liver parasitic burden (93.5%) without causing any toxicity when given orally. [Display omitted] [ABSTRACT FROM AUTHOR]

The capacity to infect and survive in a wide variety of host cells is amongst the strategies that contribute to pathogen persistence. The recent study by Karagiannis et al. presents an unbiased approach to identify infected cells in a visceral leishmaniasis (VL) infection, which revealed parasites in unexpected host cells. [ABSTRACT FROM AUTHOR]

• Visceral leishmaniasis can occur due to co-infection with several Leishmania spp. • Co-infection in visceral leishmaniasis changes our understanding of the disease. • Several trypanosomatids can be identified per sample using this protocol. • Amplicon-based next-generation sequencing is a cost-effective method in molecular epidemiological studies. Visceral leishmaniasis (VL) is a mammalian protozoal disease propagated in the Americas by female phlebotomine sandflies, mainly caused by Leishmania infantum. However, in recent years, cases of VL caused by different Leishmania species, such as L. amazonensis and L. colombiensis, have been reported in the continent. This study used an amplicon-based next-generation sequencing approach to identify VL aetiologic species using high-depth sequencing targeting a region on the Heat Shock Protein 70 gene. In this first approach, six samples from five patients diagnosed with VL were selected and analysed to identify DNA of Leishmania spp. All samples harboured DNA of L. infantum ; five samples were found to be co-infected with other Leishmania spp. or with Trypanosoma cruzi , and just one sample was mono-infected with L. infantum. This study demonstrates the usefulness of this methodology to identify trypanosomatid co-infections in clinical samples, which presents an interesting study panorama considering their biological, clinical and epidemiological implications. [Display omitted] [ABSTRACT FROM AUTHOR]

• Treatment with Nano Sbv or Anti IL-10R increase the IFN-γ producing T-CD4 lymphocytes in the initial post-treatment times. • The CVL treatment with Nano Sbv or Anti IL-10R restore the lymphoproliferative capacity in the initial post-treatment times. • Treatment does not improve the immunological, clinical and parasitological parameters at the end of the trial follow-up. This study compared the therapeutic potential of the chemotherapy using meglumine antimoniate encapsulated in a mixture of conventional and PEGylated liposomes (Nano Sbv) and immunotherapy with anti-canine IL-10 receptor-blocking monoclonal antibody (Anti IL-10R) on canine visceral leishmaniasis (CVL). Twenty mongrel dogs naturally infected by L. infantum , displaying clinical signs of visceral leishmaniasis were randomly divided in two groups. In the first one, nine dogs received six intravenous doses of a mixture of conventional and PEGylated liposomes containing meglumine antimoniate at 6.5 mg Sb/kg/dose. In the second one, eleven dogs received two intramuscular doses of 4 mg of anti-canine IL-10 receptor-blocking monoclonal antibody. The animals were evaluated before (T0) and 30, 90, and 180 days after treatments. Our major results demonstrated that both treatments were able to maintain hematological and biochemical parameters, increase circulating T lymphocytes subpopulations, increase the IFN-γ producing T-CD4 lymphocytes, restore the lymphoproliferative capacity and improve the clinical status. However, although these improvements were observed in the initial post-treatment times, they did not maintain until the end of the experimental follow-up. We believe that the use of booster doses or the association of chemotherapy and immunotherapy (immunochemotherapy) is promising to improve the effectiveness of treating CVL for improving the clinical signs and possibly reducing the parasite burden in dogs infected with Leishmania infantum. [ABSTRACT FROM AUTHOR]

[Display omitted] • Immunogenicity and prophylactic potential of rLdTCP1γ was evaluated against VL. • rLdTCP1γ caused strong LTT response & NO production in cured animal compared to SLD. • rLdTCP1γ +BCG exhibited potent prophylactic efficacy against VL infection. • rLdTCP1γ +BCG elicited both antigen specific cellular and humoral immunity. • LdTCP1γ+BCG immunization resulted in 2.5 folds increased survival of hamsters. Visceral leishmaniasis (VL) is a chronic tropical disease responsible for devastating epidemics worldwide. Though current treatment relies on drugs, the emergence of resistance, toxic side-effects, and strenuous administration has led to an ineffective remedy. Hence, vaccination remains an alternative and desirable approach for VL control. Though extensive research on anti-leishmanial vaccine candidates has been carried out in past decades, presence of an effective molecule is still missing. In the present study, we have evaluated the immunogenicity and prophylactic potential of a recombinant T-complex protein-1 gamma subunit of L. donovani (rLdTCP1γ), against VL in hamster model. The antigen exhibited in vitro stimulation of lymphoproliferative and NO response in miltefosine and amphotericin B treated hamsters depicting its immunotherapeutic/immunogenic nature. Immunization with rLdTCP1γ revealed a strong protective response against experimental VL as indicated by reduced parasite load in the spleen of immunized group compared to infected control. The immunized animals gained body weight and exhibited significant reduction in the spleen and liver weight as compared to infected controls on days 60, 90, 120 post-challenge. A substantial augmentation of cell-mediated immune response as depicted by an increased lymphocyte proliferation, nitric oxide production, DTH responses and increased levels of IgG2 was observed in rLdTCP1γ immunized hamsters. The Th1 stimulatory potential, imparted by the antigen, was found to be intensified in the presence of adjuvant Bacillus Calmette-Guérin (BCG). The efficacy was further assisted by an upregulated mRNA transcript of Th1 induced cytokines (IL-12, IFN-γ and TNFα) and downregulation of IL-4 and IL-10. The results are thus suggestive of rLdTCP1γ having the potential of a strong vaccine candidate against VL. [ABSTRACT FROM AUTHOR]

• Our research investigated the genetic variations among CL, VL, and CanL strains in northern Morocco. • Molecular and genetic analyses uncovered a complex array of genetic variants in the L. infantum and L. tropica species. The number of these variants is 7 and 9 for L. tropica and L. infantum species, respectively. • The Discriminant Analysis of Principal Components (DAPC) revealed significant genetic divergences among L. infantum strains. This suggests the influence of species origin on their genetic makeup. This study investigated nine provinces in northern Morocco and collected 275 skin scraping, 22 bone marrow aspirates, and 89 fine needle aspirations from suspected cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) patients and potentially infected dogs. Molecular analysis using ITS1 RFLP PCR and RT-PCR revealed a higher prevalence of L. infantum (66.18 %; χ2 = 28.804; df = 1; P-value = 8.01e−08) than L. tropica in skin scraping, with L. infantum being the sole causative agent for both VL and canine leishmaniasis. L. infantum was predominantly found in most provinces, while L. tropica was relatively more dominant in Taza Province. Discriminant Analysis of Principal Components (DAPC) revealed distinct clustering between L. tropica and the other three species. However, no small subset of SNPs could clearly differentiate between Infantum_CL, Infantum_VL, and CanL, as they likely share a significant genetic background. The high rate of L. infantum could be attributed to the abundance of sand fly species transmitting VL. In Taza Province, Phlebotomus sergenti, responsible for anthroponotic CL, is the most abundant species. DNA sequencing demonstrated sequence heterogeneity in L. infantum (variants 1-9) and L. tropica (variants 1-7). Phylogenetic analysis showed a distinct separation between L. tropica and L. infantum strains, with an overlap among L. infantum strains isolated from cutaneous, visceral, and canine cases, and dogs serving as the central population for L. infantum. [ABSTRACT FROM AUTHOR]

• Superparamagnetic iron oxide nanoparticles (MNPs) were synthesized for provide cheaper and high performance Leishmaniasis serodiagnosis. • Synthetic peptide it was validated in a nanomagnetic ELISA for Leishmaniasis serodiagnosis. • Nanomagnetic ELISA results has proven an advantageous technique, a less time-consuming process with optimized detection. Serodiagnosis methods have been used as platforms for diagnostic tests for many diseases. Due to magnetic nanoparticles' properties to quickly detach from an external magnetic field and particle size effects, these nanomaterials' functionalization allows the specific isolation of target analytes, enhancing accuracy parameters and reducing serodiagnosis time. Superparamagnetic iron oxide nanoparticles (MNPs) were synthesized and functionalized with polyethylene glycol (PEG) and then associated with the synthetic Leishmaniosis epitope. This nano-peptide antigen showed promising results. Regarding Tegumentary leishmaniasis diagnostic accuracy, the AUC was 0.8398 with sensibility 75% (95CI% 50.50 - 89.82) and specificity 87.50% (95CI% 71.93 - 95.03), and Visceral leishmaniasis accuracy study also present high performance, the AUC was 0.9258 with sensibility 87.50% (95CI% 63.98 - 97.78) and specificity 87.50% (95CI% 71.93 - 95.03). Our results demonstrate that the association of the antigen with MNPs accelerates and improves the diagnosis process. MNPs could be an important tool for enhancing serodiagnosis. [Display omitted] [ABSTRACT FROM AUTHOR]

• MTMR6 silencing reduces Leishmania amastigote counts in macrophages. • MTMR6 expression is higher in VL Patients but less in miltefosine-treated patients. • LPG-TLR2 interaction alters MTMR6 expression. • MTMR6siRNA lowers IL-10, but augments IL-12 and IFN-γ, expression in macrophages. Residing obligatorily as amastigotes within the mammalian macrophages, the parasite Leishmania donovani inflicts the potentially fatal, globally re-emerging disease visceral leishmaniasis (VL) by altering intracellular signaling through kinases and phosphatases. Because the phosphatases that modulate the VL outcome in humans remained unknown, we screened a human phosphatase siRNA-library for anti-leishmanial functions in THP-1, a human macrophage-like cell line. Of the 251 phosphatases, the screen identified the Ca++-activated K+-channel-associated phosphatase myotubularin-related protein-6 (MTMR6) as the only phosphatase whose silencing reduced parasite load and IL-10 production in human macrophages. Virulent, but not avirulent, L. donovani infection increased MTMR6 expression in macrophages. As virulent L. donovani parasites expressed higher lipophosphoglycan, a TLR2-ligand, we tested the effect of TLR2 stimulation or blockade on MTMR6 expression. TLR1/TLR2-ligand Pam3CSK4 enhanced, but TLR2 blockade reduced, MTMR6 expression. L. donovani infection of macrophages ex vivo increased, but miltefosine treatment reduced, MTMR6 expression. Corroboratively, compared to endemic controls, untreated VL patients had higher, but miltefosine-treated VL patients had reduced, MTMR6 expression. The phosphatase siRNA-library screening thus identified MTMR6 as the first TLR2-modulated ion channel-associated phosphatase with significant implications in VL patients and anti-leishmanial functions. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis or Kala-azar (KA) is a Vector-Borne Disease (VBD) that remains the second-largest parasitic killer across the globe (mortality rate: 75–95%). More than 60% of KA cases originate in South Asia, wherein India accounts for 2/3rd of the cases, and Bihar, a state in India, alone accounts for more than 50% of the Indian cases. Past studies suspected climate change vulnerabilities as a driving cause of KA outbreaks. The VBDs-based epidemic prediction systems have been developed to mitigate recurrent outbreaks; however, Machine Learning (ML) based approaches still need to be explored for modeling changing climate impacts on KA cases. This study, for the first time, develops a Radial Basis Function (RBF) kernel-based Support Vector Regression (SVR), hereinafter RBF-kernel-based-SVR model for the most-affected endemic districts of Bihar (northern-India), using the data from 2016 and 2021. Forward selection, backward elimination, and stepwise regression procedures were adopted while selecting influential climatic variables, followed by the k -fold cross-validation technique and, then, the RBF-kernel-based-SVR algorithm for classification. Results suggested that temperature, wind speed, rainfall, and population density significantly contributed to the KA outbreaks. This study also developed Multiple Linear Regression (MLR) and Multilayer Perceptron (MLP) models to compare SVR with other classification models. Findings indicated that the proposed RBF-kernel-based-SVR model [Correlation Coefficient (CC) = 0.82, Root-Mean-Square Error (RMSE) = 12.20, and Nash–Sutcliffe Efficiency (NSE) = 0.66] outperformed MLR (0.81, 14.20, 0.48) and MLP (0.81, 12.95, 0.61). Study recommends using the RBF-kernel-based-SVR model as a quick and efficient model capable of detecting KA cases with high predictability even under limited data availability. Such models can assist public health authorities, given monitoring KA spread, learning the climate impacts of outbreaks, and ensuring timelier health services. [ABSTRACT FROM AUTHOR]

Bats have a long evolutionary history with trypanosomatids, but the role of these flying mammals on parasite transmission cycles in urban areas, especially for Trypanosoma and Leishmania species, remains poorly known. The objective of this study was to evaluate the species richness of trypanosomatids parasitizing a bat community in Campo Grande (CG), a state capital within the Cerrado of the Brazilian Midwest. We evaluated 237 bats of 13 species by means of hemoculture and molecular detection in spleen samples. The bat community of CG appears to participate in the transmission cycles of various species of trypanosomatids. We report an overall trypanosomatid detection rate of 34.2% (n = 81), involving 11 out of 13 sampled bat species. We identified six species of trypanosomatids from 61 bats by analyzing SSU rRNA and/or kDNA: Trypanosoma cruzi DTU TcI, T. c. marinkellei , T. dionisii , Leishmania infantum , L. amazonensis , and T. janseni , with this latter being detected by hemoculture for the first time in a bat species. We also detected a Molecular Operational Taxonomic Unit, Trypanosoma sp. DID, in the phyllostomids Glossophaga soricina and Platyrrhinus lineatus. The highest trypanosomatid richness was observed for Sturnira lilium , which hosted three species: L. infantum , T. dionisii and T. janseni. Given that visceral leishmaniasis is endemic in CG, special focus should be placed on L. infantum. Moreover, L. amazonensis and T. cruzi warrant attention, since these are zoonotic parasites responsible for human cases of tegumentary leishmaniasis and Chagas disease, respectively. In this respect, we discuss how bat communities may influence the Leishmania spp. transmission in endemic areas. • The most frequent trypanosomatid were T. cruzi marinkellei and L. infantum. • We found T. cruzi DTU TcI, T. cruzi marinkellei , T. dionisii , L. infantum, L. amazonensis , and T. janseni in bats. • First detection of Trypanosoma sp. DID in bats. • First report of a bat parasitized by Trypanosoma janseni [ABSTRACT FROM AUTHOR]

• This is the first report showing that Ld CyPA induces immune escape in L. donovani. • Ld CyPA increases the survival of parasites by modifying macrophage immunological response. • Ld CyPA facilitates immune escape by blocking the MAPK signaling pathway. Visceral leishmaniasis is a neglected tropical disease affecting millions of people worldwide. Macrophages serve as the primary host cells for L. donovani , the immune response capability of these host cells is crucial for parasites' intracellular survival. L. donovani peptidyl-prolyl cis /trans isomerase Cyclophilin A (Ld CypA) is a key protein for L. donovani intracellular proliferation, while the molecular mechanism conducive to intracellular survival of parasites remains elusive. In this study, we generated a macrophage cell line overexpressing Ld CyPA to investigate its role in controlling host immunity and promoting intracellular immune escape of L. donovani. It was discovered that the overexpression of the Ld CyPA cell line regulated the host immune response following infection by downregulating the proportion of M1-type macrophages, promoting the secretion of the anti-inflammatory factor IL-4, and inhibiting the secretion of pro-inflammatory factors like IL-12, IFN-γ, TNF-α, and INOS. Transcriptome sequencing and mechanistic validation, meanwhile, demonstrated that cells overexpressing Ld CyPA controlled the immune responses that followed infection by blocking the phosphorylation of P38 and JNK1/2 proteins in the MAPK signaling pathway and simultaneously increasing the phosphorylation of ERK proteins, which helped the L. donovani escape immune recognition. Our findings thus pave the way for the development of host-directed antiparasitic drugs by illuminating the pro- Leishmania survival mechanism of L. donovani cyclophilin A and exposing a novel immune escape strategy for L. donovani that targets host cellular immune regulation. [Display omitted] [ABSTRACT FROM AUTHOR]

In Brazil, the fatality rate for visceral leishmaniasis is high, and it is important to investigate its associated factors. The aim of this study was to analyze the clinical-epidemiological profile and prognostic factors associated with death from visceral leishmaniasis (VL) in the Central-West region of Brazil, between 2010 and 2019. A study of series of VL cases was carried out using data obtained from the Sistema de Informação de Agravos de Notificação (SINAN). Multivariate logistic regression was performed to identify variables associated with deaths. Male (64.96%); age group ≤5 years (28.51%); mixed race/color (59.20%); and level of education incomplete primary education (45.16%) were the most affected. The most frequent symptoms were fever (87.65%), weakness (77.56%), enlarged spleen (70.22%) and liver (67.33%), weight loss (67.22%) and pallor (63.41%). Co-infection with HIV was observed in 15.84% of patients. The parasitological diagnosis was positive in 74.17% and the Indirect Immunofluorescence (IIF) in 82.80%. The drug most used for treatment was pentavalent antimony (41.96%). Regarding the evolution of VL, cure was recorded for 82.90% of patients and death from VL in 8.30%. Factors associated with death from VL were: age group ≥20 and <60 (OR 2.95; 95% CI 1.98–4.38) and ≥60 (OR 5.84; 95% CI 3.63–9.38), edema (OR 2.27; 95% CI 1.64–3.13), pallor (OR 1.53; 95% CI 1.06–2.20), infectious condition (OR 1.56; 95% CI 1.12–2.15) and hemorrhagic phenomena (OR 2.87; 95% CI 2.02–4.08). New studies are needed in order to better manage VL control, monitoring, prevention and primary care strategies. [Display omitted] • Mato Grosso do Sul is the state with the highest risk of visceral leishmaniasis. • Men, children aged ≤5 years, brown and with low education are the most affected. • The age groups ≥20 and < 60 and ≥ 60 are related to death by kala-azar. • Edema, pallor, infection and hemorrhage favor the outcome of death from the disease. [ABSTRACT FROM AUTHOR]

• Samples obtained by aspiration and scraping of the edge of the lesion were analyzed. • Direct parasitological exam and three methods of axenic culture in two media were assessed. • The TSTB culture of scraping lesions had the highest diagnostic sensitivity (98 %). • Diagnosis by axenic culture should be implemented whenever possible. Leishmaniases are zoonotic diseases caused by protozoa of the genus Leishmania. In Bolivia, leishmaniasis occurs mainly in the cutaneous form (CL) followed by the mucosal or mucocutaneous form (ML or MCL), grouped as tegumentary leishmaniosis (TL), while cases of visceral leishmaniasis (VL) are rare. The cases of TL are routinely diagnosed by parasitological methods: Direct Parasitological Exam (DPE) and axenic culture, the latter being performed only by specialized laboratories. The aim of the present study was to optimize the parasitological diagnosis of TL in Bolivia, using two sampling methods. Samples from 117 patients with suspected TL, obtained by aspiration (n = 121) and scraping (n = 121) of the edge of the lesion were tested by: direct parasitological exam, culture in TSTB medium, and miniculture and microculture in Schneider's medium. A positive laboratory result by any of the four techniques evaluated using either of the two sampling methods was considered the gold standard. Of the 117 suspected patients included, TL was confirmed in 96 (82 %), corresponding 79 of the confirmed cases (82.3 %) to CL and 16 (16.7 %) to ML. Parasitological techniques specificity was 100 % and their analytical sensitivity was greater with scraping samples in TSTB culture (98 %). Scraping samples in TSTB and miniculture correlated well with the reference (Cohen's kappa coefficient=0.88) and showed good reliability (Cronbach's alpha coefficient ≥0.91). Microculture provided positive results earlier than the other culture methods (mean day 4.5). By day 14, 98 % of positive cultures had been detected. Scraping sampling and miniculture were associated with higher culture contamination (6 % and 17 %, respectively). Bacterial contamination predominated, regardless of the sampling and culture method, while filamentous fungi and mixed contamination were more frequently observed in cultures from scraping samples. In conclusion: (i) scraping samples proved more suitable for the diagnosis of TL as they increased analytical sensitivity, are less traumatic for the patient and are safer for laboratory personnel than aspirates; (ii) culture, mainly in TSBT medium, should be used for the diagnosis of TL due to its high sensitivity (doubling the number of cases diagnosed by DPE) and its low cost compared to other culture media. [Display omitted] [ABSTRACT FROM AUTHOR]

Leishmaniasis is a parasitic disease caused by different Leishmania species. L. infantum is found in the Mediterranean area. It usually causes visceral or cutaneous leishmaniasis, but rarely mucosal leishmaniasis (ML). A 62-year-old man with metastatic non-small-cell lung carcinoma visited the outpatient clinic because of a painful and swollen tongue. Initially, oral candidiasis was suspected and patient was unsuccessfully treated accordingly. Subsequently, a biopsy from the tongue was taken. Histology of the tongue biopsy showed an inflammation with histiocytes and Leishmania amastigotes. Molecular analysis determined these parasites as L. donovani complex. Based on the patient's travel history, ML caused by L. infantum was diagnosed. ML is an unusual presentation of L. infantum. ML is not only caused by Leishmania species endemic in Latin America, but also should be considered in the differential diagnosis for European patients. A biopsy of the affected location is needed to confirm the diagnosis. [ABSTRACT FROM AUTHOR]

[Display omitted] • Phlebotomus argentipes is a sole vector of Leishmania donovani in the Indian subcontinent. • 40% of humans in the study area have IgG antibodies against P. argentipes saliva. • A correlation was found between IgG responses against P. argentipes saliva and rPagSP06. • rPagSP06 is a valid antigen to measure human exposure to P. argentipes. Phlebotomus argentipes is a predominant vector of Leishmania donovani , the protozoan parasite causing visceral leishmaniasis in the Indian subcontinent. In hosts bitten by P. argentipes , sand fly saliva elicits the production of specific anti-salivary protein antibodies. Here, we have utilised these antibodies as markers of human exposure to P. argentipes in a visceral leishmaniasis endemic area in Pabna district, Bangladesh. The use of whole salivary gland homogenate as an antigen to detect these antibodies has several limitations, therefore it is being superseded by the use of specific recombinant salivary proteins. We have identified three major P. argentipes salivary antigenic proteins recognised by sera of bitten humans, expressed them in a recombinant form (rPagSP04, rPagSP05 and rPagSP06) and tested their applicability in ELISA and immunoblot. One of them, PpSP32-like protein rPagSP06, was identified as the most promising antigen, showing highest resemblance and correlation with the IgG response to P. argentipes salivary gland homogenate. Furthermore, we have validated the applicability of rPagSP06 in a large cohort of 585 individuals and obtained a high correlation coefficient for anti-rPagSP06 and anti- P. argentipes saliva IgG responses. The anti-rPagSP06 and anti- P. argentipes salivary gland homogenate IgG responses followed a similar right-skewed distribution. This is the first report of screening human sera for anti- P. argentipes saliva antibodies using recombinant salivary protein. The rPagSP06 was proven to be a valid antigen for screening human sera for exposure to P. argentipes bites in a visceral leishmaniasis endemic area. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a neglected parasitic disease for which the conventional treatment can be considered inefficient and extremely aggressive, generating several and severe side effects. Therefore, the discovery of new drug candidates is important for the improvement in the quality of life of patients. Previously, we reported the promising results of isopentyl caffeate (ICaf) against Leishmania chagasi (agent of visceral leishmaniasis) and Leishmania amazonensis (agent of cutaneous leishmaniasis) promastigotes, displaying IC 50 of 1.56 and 1.71 μM, respectively. Herein, we aimed to decipher the mechanisms of anti- Leishmania action of ICaf. Light and scanning electron microscopy assays showed relevant morphological changes in promastigotes when treated with ICaf, including rounding of the parasite body, shortening of the flagellum, blebs on the plasma membrane and cellular aggregation. The parasite mitochondrion was targeted by ICaf, resulting in a significant reduction in its metabolic activity and electric membrane potential followed by an increase in the production of reactive oxygen species, which culminated in the loss of plasma membrane integrity and parasite death. Relevantly, ICaf also had a potent anti-amastigote action. The IC 50 values calculated for intracellular amastigotes of L. amazonensis were 3.27, 1.60 and 1.52 μM, while for L. chagasi the values were 2.48, 1.84 and 1.60 μM, respectively, after treating the infected macrophages with ICaf for 24, 48 and 72 h. ICaf was well tolerated by THP-1 macrophages, which gave rise to excellent selectivity indexes considering both Leishmania species. The current results suggest that ICaf may emerge as a chemotherapeutic alternative for the treatment of leishmaniasis. • Treatment of leishmania with conventional drugs is regarded as inefficient and extremely aggressive. • Isopentyl caffeate was well tolerated by THP-1 macrophages with high selectivity. • Isopentyl caffeate may emerge as a chemotherapeutic treatment alternative. [ABSTRACT FROM AUTHOR]

• L. infantum showed a lower percentage of infection in presence of anti-canine IL-10 receptor-blocking monoclonal antibody. • The presence of Bloq IL-10R enhanced T-CD4 and CD8 proliferative response in PBMC of dogs infected by L. infantum. • The presence of Bloq IL-10R altered the cytokine synthesis with reduction of CD4+IL-4+ cells and increased of CD8+IFN-γ+. An important strategy to reduce the risk of visceral leishmaniasis (VL) in humans is to control the infection and disease progression in dogs, the domestic reservoir of Leishmania infantum parasites. Certain therapeutic strategies that modulate the host immune response show great potential for the treatment of experimental VL, restoring the impaired effector functions or decreasing host excessive responses. It is known that the overproduction of interleukin-10 (IL-10) promotes parasite replication and disease progression in human VL as well as in canine visceral leishmaniasis (CVL). Thus, in the present study we investigated the potential of the anti-canine IL-10 receptor-blocking monoclonal antibody (Bloq IL-10R) to control and reduce in vitro infectivity of L. infantum and improve the ability of PBMC isolated from VL dogs to alter the lymphoproliferative response and intracytoplasmic cytokines. Overall, GFP+ Leishmania showed lower capacity of in vitro infectivity in the presence of Bloq IL-10R. Moreover, addition of Bloq IL-10R in cultured PBMC enhanced T-CD4 and CD8 proliferative response and altered the intracytoplasmic cytokine synthesis, reducing CD4+IL-4+ cells and increasing CD8+IFN-γ+ cells after specific antigen stimulation in PBMC of dogs. Furthermore, we observed an increase of TNF-α levels in supernatant of cultured PBMC under IL-10R neutralizing conditions. Together, our findings are encouraging and reaffirm an important factor that could influence the effectiveness of immune modulation in dogs with VL and suggest that blocking IL-10R activity has the potential to be a useful approach to CVL treatment. [ABSTRACT FROM AUTHOR]

In Brazil, canine visceral leishmaniasis is an important public health problem due to its alarming growth. The high prevalence of infected dogs reinforces the need for a vaccine for use in prophylactic vaccination campaigns. In the present study, we evaluate the immunogenicity and protection of the best dose of Chimera A selected through the screening of cytokines production important in disease. BALB/c mice were vaccinated subcutaneously with three doses and challenged intravenously with 1 × 107 L. infantum promastigotes. Spleen samples were collected to assess the intracellular cytokine profile production, T cell proliferation and parasite load. At first, three different doses of Chimera A (5 μg, 10 μg and 20 μg) were evaluated through the production of IFN-γ and IL-10 cytokines. Since the dose of 20 μg showed the best results, it was chosen to continue the study. Secondarily, Chimera A at dose of 20 μg was formulated with Saponin plus Monophosphoryl lipid A. Vaccination with Chimera A alone and formulated with SM adjuvant system was able to increase the percentage of the proliferation of specific T lymphocytes and stimulated a Th1 response with increased levels of IFN-γ, TNF-α and IL-2, and decreased of IL-4 and IL-10. The vaccine efficacy through real-time PCR demonstrated a reduction in the splenic parasite load in animals that received Chimera A formulated with the SM adjuvant system (92%). Additionally, we observed increased levels of nitric oxide in stimulated-culture supernatants. The Chimera A formulated with the SM adjuvant system was potentially immunogenic, being able to induce immunoprotective mechanisms and reduce parasite load. Therefore, the use of T-cell multi-epitope vaccine is promising against visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

• Leishmania infantum chagasi LPG3 protein is immunogenic in BALB/c model; • LPG3 plus IFA partially protects BALB/c mice against L. infantum chagasi infection; • LPG3 plus saponin induces 98% reduction of BALB/c splenic and hepatic parasitism; • Results using rLPG3 are similar to the results using native LPG3. Visceral leishmaniasis (VL) is a serious neglected tropical disease that affects humans and dogs in urban areas. There are no vaccines against human VL, and few licensed canine VL vaccines are currently available, which instigates the search for new antigens and vaccine formulations with prophylactic potential against VL in these hosts. In this study, we evaluated the immunization using the native and recombinant Leishmania infantum chagasi (L. chagasi) lipophosphoglycan-3 (LPG3) and the adjuvants saponin (SAP) and incomplete Freund adjuvant (IFA) against L. chagasi infection in BALB/c mice. The native LPG3 vaccine was immunogenic, inducing splenic IFN-γ and IL-10 production, and mixed Th1/Th2 response when associated with IFA. However, only mice vaccinated with LPG3-IFA presented a reduction in the splenic parasite load (96% in comparison to the PBS control group), but without a significant reduction in the hepatic parasitism. On the other hand, mice immunized with the LPG3-SAP vaccine presented a reduction of approximately 98% in both splenic and hepatic parasite load, accompanied by a Th1/Th17 response and IL-10 production by L. chagasi antigen (Ag Lc)-stimulated splenic cells. Importantly, vaccination with recombinant LPG3 (rLPG3)-SAP presented similar results to the native LPG3-SAP vaccine. Therefore, the rLPG3-SAP vaccine is qualified to be used in future tests in canine and human models, considering the technical and economic advantages of the recombinant protein production compared to the native protein and the results obtained in the murine model. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is the most lethal among all leishmaniasis diseases and remains categorized as a neglected tropical disease (NTD). This study aimed to develop a peptide-based multi-epitope vaccine construct against VL using immunoinformatics methodologies. To achieve this, four distinct proteins were screened to identify peptides consisting of 9–15 amino acids with high binding affinity to toll-like receptors (TLRs), strong antigenicity, low allergenicity, and minimal toxicity. The resulting multi-epitope vaccine construct was fused in a tandem arrangement with appropriate linker peptides and exhibited superior properties related to cytotoxic T lymphocytes (CTLs), helper T lymphocytes (HTLs), and B-cell epitopes. Subsequently, a three-dimensional (3D) model of the vaccine construct was generated, refined, and validated for structural stability and immune response capabilities. Molecular docking and simulations confirmed the vaccine construct's stability and binding affinities with TLRs, with TLR4 displaying the highest binding affinity, followed by TLR2 and TLR3. Additionally, simulations predicted robust cellular and humoral antibody-mediated immune responses elicited by the designed vaccine construct. Notably, this vaccine construct includes proteins from various pathways of Leishmania donovani (LD), which have not been previously utilized in VL vaccine design. Thus, this study opens new avenues for the development of vaccines against diverse protozoan diseases. [ABSTRACT FROM AUTHOR]

Leishmania donovani is the causative unicellular parasite for visceral leishmaniasis (VL); and Fe S proteins are likely to be very essential for their survival and viability. Cytosolic Fe S cluster assembly (CIA) machinery is one of the four systems for the biosynthesis and transfer of Fe S clusters among eukaryotes; Cfd1 and Nbp35 are the scaffold components for cytosolic Fe S cluster biogenesis. We investigated the role of CIA machinery components and purified Cfd1 and Nbp35 proteins of L. donovani. We also investigated the interactive nature between LdCfd1 and LdNbp35 proteins by in silico analysis, in vitro co-purification, pull down assays along with in vivo immuno-precipitation; which inferred that both LdCfd1 and LdNbp35 proteins are interacting with each other. Thus, our collective data revealed the interaction between these two proteins which forms a stable complex that can be attributed to the cellular process of Fe S clusters biogenesis, and transfer to target apo-proteins of L. donovani. The expression of Cfd1 and Nbp35 proteins in Amp B resistant parasites is up-regulated leading to increased amount of Fe S proteins. Hence, it favors increased tolerance towards ROS level, which helps parasites survival under drug pressure contributing in Amphotericin B resistance. [ABSTRACT FROM AUTHOR]

• The ongoing activities of NKEP are focused on endemic areas in Bangladesh. • Transmission of the disease is likely to occur in non-programmatic areas. • Epidemiological, serological, and entomological investigation is necessary in non-programmatic areas. • Asymptomatic carriers and the disease vector have been found in these areas. • NKEP should continue program activities in the newly identified foci to prevent disease transmission. The study aimed to explore epidemiological, serological, and entomological aspects of visceral leishmaniasis (VL) in suspected new VL foci and assess the knowledge, attitude, and practices of the community living in the alleged new VL foci. The study investigated new visceral leishmaniasis (VL) cases reported between 2019 and 2020 in four sub-districts (Dharmapasha, Hakimpur, Islampur and Savar) where we tested 560 members using the rK39 rapid test and conducted vector collections in six neighbouring houses of the index cases to assess sandfly density and distribution, examined sandflies' infection, and determined the spatial relationship with VL infection. Furthermore, we highlighted the importance of early detection, and community awareness in controlling the spread of the disease. The study screened 1078 people from 231 households in the four sub-districts for fever, history of visceral leishmaniasis (VL), and PKDL-like skin lesions. Among sub-districts, positivity rate for rK39 rapid test was highest (3.5 %) in Savar. Sandflies were present across all areas except in Dharmapasha, but all 21 collected female P. argentipes sandflies were negative for Leishmania parasite DNA. We found one person from Islampur with a history of VL, and one from Islampur and another one from Savar had PKDL. After the awareness intervention, more people became familiar with VL infection (91.2 %), and their knowledge concerning sandflies being the vector of the disease and the risk of having VL increased significantly (30.1 %). The study found no active case in the suspected new foci, but some asymptomatic individuals were present. As sandfly vectors exist in these areas, the National Kala-azar Elimination Programme (NKEP) should consider these areas as kala-azar endemic and initiate control activities as per national guidelines. [ABSTRACT FROM AUTHOR]

• Transcriptional analysis allows the identification of large-scale changes in mice spleen. • Immune and metabolic deregulation of genes during early Leishmania infantum infection. • Very early activation of immune response turns ineffective at day 10 post-infection. • Downregulation of metabolic markers hampers parasitic control in infected spleen tissue. • Il21 identified as a potential early biomarker of visceral leishmaniasis in mice spleen. Transcriptional analysis is a useful approximation towards the identification of global changes in host-pathogen interaction, in order to elucidate tissue-specific immune responses that drive the immunopathology of the disease. For this purpose, expression of 223 genes involved in innate and adaptive immune response, lipid metabolism, prostaglandin synthesis, C-type lectin receptors and MAPK signaling pathway, among other processes, were analyzed during the early infection in spleens of BALB/c mice infected by Leishmania infantum. Our results highlight the activation of immune responses in spleen tissue as early as 1 day p.i., but a mixed pro-inflammatory and regulatory response at day 10 p.i., failing to induce an effective response towards control of Leishmania infection in the spleen. This ineffective response is coupled to downregulation of metabolic markers relevant for pathways related to icosanoid biosynthesis , adipocytokine signaling or HIF-1 signaling, among others. Interestingly, the over-representation of processes related to immune response, revealed Il21 as a potential early biomarker of L. infantum infection in the spleen. These results provide insights into the relationships between immune and metabolic responses at transcriptional level during the first days of infection in the L. infantum -BALB/c experimental model, revealing the deregulation of many important pathways and processes crucial for parasitic control in infected tissues. [ABSTRACT FROM AUTHOR]