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Introduction: Hemophagocytic lymphohistiocytosis characterized by hemophagocytosis leading to uncontrolled inflammation; the most common etiology in secondary cases of hemophagocytic lymphohistiocytosis is viral infections, especially Epstein–Barr virus. Visceral leishmaniasis is a vectorborne protozoal disease caused by Leishmania donovani complex. It is common in tropical and subtropical regions, with 50,000–90,000 new cases annually. Case presentation: A 15-month-old Arab female was admitted to our hospital with 15 days of fever and decreased weight. On clinical examination, she had a markedly enlarged liver and spleen that were palpable 4 cm and 6 cm below the costal margin, respectively. The peripheral blood smear showed hypochromic microcytic anemia, poikilocytosis, reactive lymphocytosis, and mild thrombocytopenia. Bone marrow aspiration did not show malignancy or any other pathological findings. The patient was put on antibiotic therapy without improvement. Repeated bone marrow aspiration showed erythrophagocytosis; intracellular small round organisms looked like the amastigote form of Leishmania (Donovan bodies) with no evidence of malignancies. Her lab values showed ferritin greater than 500 ug/L, pancytopenia, and hypertriglyceridemia. The patient was diagnosed with hemophagocytic lymphohistiocytosis secondary to visceral leishmaniasis. Conclusion: Hemophagocytic lymphohistiocytosis secondary to visceral leishmaniasis is an extensively rare phenomenon in the medical literature that causes challenges in diagnosis and management. Steroids should be used wisely to not cover the symptoms of infections or malignancy, and amphotericin B resistance should be kept in mind in unresponsive Leishmania cases. [ABSTRACT FROM AUTHOR]

Human visceral leishmaniasis (VL) is an endemic disease in eastern Sudan, particularly in Al Gadarif State, causing significant morbidity and fatality rates. This disease is induced by Leishmania donovani and Leishmania infantum. Several studies have suggested that dogs (Canis familiaris), which are known for being the primary reservoir hosts of L. infantum (causing canine leishmaniasis), may play a vital role in transmitting human VL caused by L. donovani. This study aimed to determine the prevalence of leishmaniasis in general and L. donovani, particularly in dogs, and to investigate the potential risk factors associated with the disease in Al Gadarif State. Blood samples were collected from 151 dogs from five localities and examined using Giemsa-stained blood smears and PCR. The overall prevalence was 44.4% based on microscopic examination. Significant variation in the prevalence of leishmaniasis was observed among the localities (p = 0.000), where the highest prevalence was reported in Algurreisha (85%) and the lowest was observed in Alhawata (4.2%). Dog type was another risk factor that was shown to be significantly associated with infection (p = 0.041). The highest prevalence was reported in household dogs (47.7%) compared to stray dogs (23.8%). The analysis revealed that factors such as the use of dogs, sex, and age were not statistically linked to the disease. However, among household dogs, the highest prevalence was reported in pet dogs (59.7%), males (49.1%) compared to females (32.6%), and young dogs (50%) compared to older dogs (40.9%). All blood samples tested negative for L. donovani by PCR, thus requiring further investigation to identify the causative species in dogs and their possible roles in the epidemiology of zoonotic leishmaniasis in Sudan. [ABSTRACT FROM AUTHOR]

Concerns regarding toxicity and resistance of current drugs in visceral leishmaniasis have been reported. Antimicrobial peptides are considered to be promising candidates and among them human cathelicidin hCAP18/LL-37 showed significant parasite killing on drug-sensitive and resistant Leishmania promastigotes, in addition to its apoptosis-inducing role. Administration of hCAP18/LL-37 to infected macrophages also decreased parasite survival and increased the host favorable cytokine interleukin 12. However, 1,25-dihydroxyvitamin D3 (vitamin D3)-induced endogenous hCAP18/LL-37 production was hampered in infected THP-1 cells. Infection also suppressed the vitamin D3 receptor (VDR), transcription factor of hCAP18/LL-37. cAMP response element modulator (CREM), the repressor of VDR, was induced in infection, resulting in suppression of both VDR and cathelicidin expression. PGE2/cAMP/PKA axis was found to regulate CREM induction during infection and silencing CREM in infected cells and BALB/c mice led to decreased parasite survival. This study documents the antileishmanial potential of cathelicidin and further identifies CREM as a repressor of cathelicidin in Leishmania infection. [ABSTRACT FROM AUTHOR]

Liposomal amphotericin B is an important frontline drug for the treatment of visceral leishmaniasis, a neglected disease of poverty. The mechanism of action of amphotericin B (AmB) is thought to involve interaction with ergosterol and other ergostane sterols, resulting in disruption of the integrity and key functions of the plasma membrane. Emergence of clinically refractory isolates of L. donovani and L. infantum is an ongoing issue and knowledge of potential resistance mechanisms can help to alleviate this problem. Here we report the characterisation of four independently selected L. donovani clones that are resistant to AmB. Whole genome sequencing revealed that in three of the moderately resistant clones, resistance was due solely to the deletion of a gene encoding C24-sterol methyltransferase (SMT1). The fourth, hyper-resistant resistant clone (>60-fold) was found to have a 24 bp deletion in both alleles of a gene encoding a putative cytochrome P450 reductase (P450R1). Metabolic profiling indicated these parasites were virtually devoid of ergosterol (0.2% versus 18% of total sterols in wild-type) and had a marked accumulation of 14-methylfecosterol (75% versus 0.1% of total sterols in wild-type) and other 14-alpha methylcholestanes. These are substrates for sterol 14-alpha demethylase (CYP51) suggesting that this enzyme may be a bona fide P450R specifically involved in electron transfer from NADPH to CYP51 during catalysis. Deletion of P450R1 in wild-type cells phenocopied the metabolic changes observed in our AmB hyper-resistant clone as well as in CYP51 nulls. Likewise, addition of a wild type P450R1 gene restored sterol profiles to wild type. Our studies indicate that P450R1 is essential for L. donovani amastigote viability, thus loss of this gene is unlikely to be a driver of clinical resistance. Nevertheless, investigating the mechanisms underpinning AmB resistance in these cells provided insights that refine our understanding of the L. donovani sterol biosynthetic pathway. Author summary: The antifungal drug, amphotericin B, is also used in the treatment of visceral leishmaniasis, a potentially lethal parasitic disease infecting the specialised immune cells (macrophages) in the liver, spleen, and bone marrow. Treatment failures due to emerging drug resistance are a significant concern. Using a combination of genetic and biochemical approaches, we have confirmed the mechanisms by which these parasites become less sensitive to treatment with amphotericin B. In addition, we have identified a novel mechanism involving loss of a key enzyme (cytochrome P450 reductase 1) in the biosynthetic pathway to ergosterol, an important lipid component of the parasite's plasma membrane. These studies increase our fundamental understanding of this important metabolic pathway and provide information that may be exploited to develop novel therapeutic strategies to combat this killer disease. [ABSTRACT FROM AUTHOR]

In an area endemic with Indian visceral leishmaniasis (VL), we performed direct xenodiagnosis to evaluate the transmission of Leishmania donovani from patients with VL–human immunodeficiency virus (HIV) coinfection to the vector sandflies, Phlebotomus argentipes. Fourteen patients with confirmed VL-HIV coinfection, with a median parasitemia of 42 205 parasite genome/mL of blood, were exposed to 732 laboratory-reared pathogen-free female P argentipes sandflies on their lower arms and legs. Microscopy revealed that 16.66% (122/732) of blood-fed flies were xenodiagnosis positive. Notably, 93% (13/14) of the VL-HIV group infected the flies, as confirmed by quantitative polymerase chain reaction and/or microscopy, and were 3 times more infectious than those who had VL without HIV. [ABSTRACT FROM AUTHOR]

Leishmania donovani surface glycoprotein 63 (GP63) is a major virulence factor involved in parasite escape and immune evasion. In this study, we generated virus-like particles (VLPs) expressing L. donovani GP63 using the baculovirus expression system. Mice were intramuscularly immunized with GP63-VLPs and challenged with L. donovani promastigotes. GP63-VLP immunization elicited higher levels of L. donovani antigen-specific serum antibodies and enhanced splenic B cell, germinal center B cell, CD4+, and CD8+ T cell responses compared to unimmunized controls. GP63-VLPs inhibited the influx of pro-inflammatory cytokines IFN-γ and IL-6 in the livers, as well as thwarting the development of splenomegaly in immunized mice. Upon L. donovani challenge infection, a drastic reduction in splenic parasite burden was observed in VLP-immunized mice. These results indicate that GP63-VLPs immunization conferred protection against L. donovani challenge infection by inducing humoral and cellular immunity in mice. Author summary: Visceral leishmaniasis is a neglected tropical disease that can be fatal in humans if left untreated. Unfortunately, despite the urgent need, an efficacious vaccine for this parasitic disease continues to remain under development. Though challenging, we attempted to develop an experimental leishmaniasis vaccine using the highly immunogenic virus-like particle (VLP) platform that expressed the glycoprotein 63 antigen of Leishmania donovani, a causative agent of visceral leishmaniasis. We used this vaccine to immunize mice and after two immunizations, mice were challenge infected with L. donovani to assess vaccine-induced protection. Our results revealed that compared to the unimmunized control mice, mice immunized with the experimental vaccines were well-protected against developing visceral leishmaniasis. Specifically, VLP vaccine immunization ensured that immune responses necessary for protection were induced to high levels and this contributed to suppressing leishmaniasis-associated pathologies as well as the parasite propagation in mice. Overall, we demonstrated that this VLP-based vaccine can elicit adequate protection against visceral leishmaniasis and propose further research to enhance and optimize its efficacy. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is an infectious disease caused by protozoa, Leishmania. During infection, the cell-mediated immunity of host gets impeded, so immunomodulation using natural products can be considered as an effective therapeutic strategy. The present study deals with the investigation of antileishmanial and immunostimulatory potential of hydro-ethanolic root extract of Rhodiola imbricata (HERERI) in inbred BALB/c mice. The secondary metabolites were analyzed quantitatively. To study the effect of HERERI on promastigotes, assays including inhibitory concentration (IC50) by trypan blue staining, cell cycle arrest by FACS were performed. The splenic parasite load was monitored after 14 days of treatment in BALB/c mice (500 and 1000 mg kg−1 b.wt.). The immunological responses after treatment were recorded in terms of DTH (delayed type of hypersensitivity) reactions, different T lymphocyte's subtypes and amounts of Th1/2 cytokines. Biochemical assays to analyze the function of liver and kidney were also done along with the histological studies. HERERI contains a considerable amount of flavonoids, phenols and terpenoids. The IC50 of 24.84 ± 0.52 and 46.65 ± 1.90 µg mL−1 against sensitive and resistant strain was obtained, respectively. It also arrested the cell cycle of promastigotes at sub G0-G1 phase. HERERI significantly decreased parasite load and up-scaled the DTH responses. It also polarized the Th1 dominating immune status by upregulation of IL-12, IFN-γ, and TNF-α cytokines and CD4+, CD8+ T cells. It caused no toxicity as biochemical tests and the histological sections were normal as compared to infected animals. Overall R. imbricata rescued mice from VL infection through immunostimulation. [ABSTRACT FROM AUTHOR]

Background: Diagnosis of visceral leishmaniasis (VL) in resource-limited endemic regions is currently based on serological testing with rK39 immunochromatographic tests (ICTs). However, rK39 ICT frequently has suboptimal diagnostic accuracy. Furthermore, treatment monitoring and detection of VL relapses is reliant on insensitive and highly invasive tissue aspirate microscopy. Miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative and user-friendly molecular tool which does not require DNA extraction and uses a lateral flow strip for result read-out. This assay could be an interesting candidate for more reliable VL diagnosis and safer test of cure at the point of care. Methodology/Principle findings: The performance of mini-dbPCR-NALFIA for diagnosis of VL in blood was assessed in a laboratory evaluation and compared with the accuracy of rK39 ICTs Kalazar Detect in Spain and IT LEISH in East Africa. Limit of detection of mini-dbPCR-NALFIA was 650 and 500 parasites per mL of blood for Leishmania donovani and Leishmania infantum, respectively. In 146 blood samples from VL-suspected patients from Spain, mini-dbPCR-NALFIA had a sensitivity of 95.8% and specificity 97.2%, while Kalazar Detect had a sensitivity of 71.2% and specificity of 94.5%, compared to a nested PCR reference. For a sample set from 58 VL patients, 10 malaria patients and 68 healthy controls from Ethiopia and Kenya, mini-dbPCR-NALFIA had a pooled sensitivity of 87.9% and pooled specificity of 100% using quantitative PCR as reference standard. IT LEISH sensitivity and specificity in the East African samples were 87.9% and 97.4%, respectively. Conclusions/Significance: Mini-dbPCR-NALFIA is a promising tool for simplified molecular diagnosis of VL and follow-up of treated patients in blood samples. Future studies should evaluate its use in endemic, resource-limited settings, where mini-dbPCR-NALFIA may provide an accurate and versatile alternative to rK39 ICTs and aspirate microscopy. Author summary: Visceral leishmaniasis (VL) is a severe infectious disease caused by unicellular parasites of the Leishmania donovani complex. As VL is fatal when left untreated, early and confirmatory laboratory diagnosis is crucial for adequate patient management. However, currently available serological assays, such as the rK39 rapid diagnostic test, fall short in terms of sensitivity and cannot be used for detection of relapsing infections. Therefore, we evaluated an innovative and user-friendly diagnostic assay called mini-dbPCR-NALFIA, which is based on the direct detection of Leishmania kinetoplast DNA in human blood samples. We found that mini-dbPCR-NALFIA can detect down to 500 parasites per millilitre of blood, which is lower than the parasite loads generally seen in untreated VL patients. Furthermore, we assessed the accuracy of mini-dbPCR-NALFIA using a set of Spanish and East-African samples from VL patients and control groups. In the Spanish samples, sensitivity of mini-dbPCR-NALFIA was very high and somewhat better than in the East-African samples. The specificity of mini-dbPCR-NALFIA was excellent in both sample sets. In conclusion, considering its robust performance and simplicity, the mini-dbPCR-NALFIA is a promising diagnostic assay that has the potential to improve VL diagnosis and follow-up of treated patients, especially in resource-limited situations. [ABSTRACT FROM AUTHOR]

The treatment for visceral leishmaniasis (VL) causes toxicity in patients, entails high cost and/or leads to the emergence of resistant strains. No human vaccine exists, and diagnosis presents problems related to the sensitivity or specificity of the tests. Here, we tested two phage clones, B1 and D11, which were shown to be protective against Leishmania infantum infection in a murine model as immunotherapeutics to treat mice infected with this parasite species. The phages were used alone or with amphotericin B (AmpB), while other mice received saline, AmpB, a wild‐type phage (WTP) or WTP/AmpB. Results showed that the B1/AmpB and D11/AmpB combinations induced polarised Th1‐type cellular and humoral responses, which were primed by high levels of parasite‐specific IFN‐γ, IL‐12, TNF‐α, nitrite and IgG2a antibodies, which reflected in significant reductions in the parasite load in distinct organs of the animals when analyses were performed 1 and 30 days after the treatments. Reduced organic toxicity was also found in these animals, as compared with the controls. In conclusion, preliminary data suggest the potential of the B1/AmpB and D11/AmpB combinations as immunotherapeutics against L. infantum infection. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a vector‐borne parasitic disease that possesses high morbidity and a variety of clinical manifestations. Of all the clinical manifestations, visceral leishmaniasis (VL) is the deadliest and its etiological organisms are mainly Leishmania infantum (L. infantum) and Leishmania donovani (L. donovani). In the tropical and subtropical regions, L. infantum is the main causative agent of VL, and it creates a favorable biological milieu in the host through the multifaceted action of its trypanothione reductase (TryR), which mediates the reduction of oxidized trypanothione to its reduced form, leading to the replenishment of the antioxidant capacity of the parasite and detoxifying the reactive oxygen and nitrogen species imposed by the host's immune system. The distinct structural and functional differences of this enzyme compared to its human homologs and its pivotal role in the survival of the parasite have rendered it an attractive target for drug discovery odysseys. Consequently, this study aims to identify potential inhibitors of TryR of L. infantum from Allium sativum (A. sativum) using molecular modeling techniques. The identified compounds were first screened using molecular docking, after which the pharmacokinetics properties of the top‐scoring compounds were further evaluated. Further evaluation of the stability of the complexes of the lead compounds with TryR revealed their relative stability over 100 ns simulation period. Ultimately, the lead compounds namely beta‐sitosterol, (E)‐alpha‐bisabolene, and agapanthagenin were found to possess suitable properties based on the results of this study and can serve as inhibitors of TryR of L. infantum upon further in vitro and in vivo study. [ABSTRACT FROM AUTHOR]

Leishmania donovani are intracellular, human blood parasites that cause visceral leishmaniasis or kala-azar. Cell-penetrating peptides (CPPs) have been shown to modulate intracellular processes and cargo delivery, whereas host defense peptides (HDPs) promote proliferation of both naïve and antigen activated CD4+ T-cells. We report newly designed tripeptides that were able to trigger proinflammatory cytokine (IL-12 and IFN-γ) secretion by CD4+CD44+ T-cells in response to Leishmania donovani infection. These peptides can be used to induce antigen specific TH1 responses to combat obstacles of cytotoxicity and drug resistance associated with current anti-leishmanial drugs. Furthermore, these peptides can also be used as adjuvants to develop an effective immunoprophylactic approach for immunity restoration against visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL), often referred to as kala-azar, is quite rare in developed countries during pregnancy. Only few studies have evaluated its impact on perinatal outcome. It is caused primarily by Leishmania donovani or Leishmania infantum and presents with a wide spectrum of clinical manifestations from cutaneous ulcers to multisystem disease. Differential diagnosis is challenging as symptoms and signs are insidious, mimicking other diseases. Misdiagnosis can result in severe adverse perinatal outcomes, even maternal/neonatal death. Early treatment with liposomal amphotericin-B (LAmB) is currently the first choice with adequate effectiveness. We report a rare case of VL in a twin pregnancy with onset at the second trimester, presenting with periodic fever with rigors, right flank pain, and gradual dysregulation of all three cell lines. The positive rK39 enzyme-linked immunosorbent assay test confirmed the diagnosis. Treatment with LAmB resulted in clinical improvement within 48 h and in the delivery of two late-preterm healthy neonates with no symptoms or signs of vertical transmission. The one-year follow-up, of the mother and the neonates, was negative for recurrence. To our knowledge, this is the first reported case of VL in a twin pregnancy, and consequently treatment and perinatal outcome are of great importance. [ABSTRACT FROM AUTHOR]

In Nepal, visceral leishmaniasis (VL) has been targeted for elimination as a public health problem by 2026. Recently, increasing numbers of VL cases have been reported from districts of doubtful endemicity including hills and mountains, threatening the ongoing VL elimination program in Nepal. We conducted a multi-disciplinary, descriptive cross-sectional survey to assess the local transmission of Leishmania donovani in seven such districts situated at altitudes of up to 1,764 meters in western Nepal from March to December 2019. House-to-house surveys were performed for socio-demographic data and data on past and current VL cases. Venous blood was collected from all consenting individuals aged ≥2 years and tested with the rK39 RDT. Blood samples were also tested with direct agglutination test, and a titer of ≥1:1600 was taken as a marker of infection. A Leishmania donovani species-specific PCR (SSU-rDNA) was performed for parasite species confirmation. We also captured sand flies using CDC light traps and mouth aspirators. The house-to-house surveys documented 28 past and six new VL cases of which 82% (28/34) were without travel exposure. Overall, 4.1% (54/1320) of healthy participants tested positive for L. donovani on at least one serological or molecular test. Among asymptomatic individuals, 17% (9/54) were household contacts of past VL cases, compared to 0.5% (6/1266) among non-infected individuals. Phlebotomus argentipes, the vector of L. donovani, was found in all districts except in Bajura. L. donovani was confirmed in two asymptomatic individuals and one pool of sand flies of Phlebotomus (Adlerius) sp. We found epidemiological and entomological evidence for local transmission of L. donovani in areas previously considered as non-endemic for VL. The national VL elimination program should revise the endemicity status of these districts and extend surveillance and control activities to curb further transmission of the disease. [ABSTRACT FROM AUTHOR]

Lipids stored in lipid-bodies (LBs) in host cells are potential sources of fatty acids for pathogens. However, the mechanism of recruitment of LBs from the host cells by pathogens to acquire fatty acids is not known. Here, we have found that Leishmania specifically upregulates the expression of host Rab18 and its GEF, TRAPPC9 by downregulating the expression of miR-1914-3p by reducing the level of Dicer in macrophages via their metalloprotease gp63. Our results also show that miR-1914-3p negatively regulates the expression of Rab18 and its GEF in cells. Subsequently, Leishmania containing parasitophorous vacuoles (Ld-PVs) recruit and retain host Rab18 and TRAPPC9. Leishmania infection also induces LB biogenesis in host cells and recruits LBs on Ld-PVs and acquires FLC12-labeled fatty acids from LBs. Moreover, overexpression of miR-1914-3p in macrophages significantly inhibits the recruitment of LBs and thereby suppresses the multiplication of parasites in macrophages as parasites are unable to acquire fatty acids. These results demonstrate a novel mechanism how Leishmania acquire fatty acids from LBs for their growth in macrophages. Author summary: Leishmania donovani which causes fatal disease known as visceral leishmaniasis, is dependent on its host to scavenge fatty acids for its survival. But, how Leishmania get fatty acids from host macrophages is not known. Lipid bodies are unique organelles that contain neutral lipids and fatty acids, therefore, LBs in macrophages could be the potential source of fatty acids for parasites. We have found that Leishmania upregulate the expression of host Rab18 and its GEF by downregulating the expression of miR-1914-3p in infected macrophages via the Ld-gp63-mediated inhibition of dicer expression. As biogenesis of LBs is shown to be regulated by Rab18, we have found that Leishmania infection induces LB biogenesis in macrophages and subsequently recruits LBs along with Rab18 and its GEF on Leishmania containing parasitophorous vacuoles. Finally, parasites-containing vacuoles acquire FLC12-labeled fatty acids from LBs to meet their nutritional requirement. We have also shown that overexpression of miR-1914-3p in macrophages significantly inhibits the recruitment of LBs and thereby suppresses the growth of parasites in macrophages as parasites are unable to acquire nutrients from LBs. These results demonstrate how Leishmania acquire nutrients like fatty acids from LBs for their proliferation in macrophages. [ABSTRACT FROM AUTHOR]

Background: CD4+ T cells play a central role in control of L. donovani infection, through IFN-γ production required for activation of macrophages and killing of intracellular parasites. Impaired control of parasites can in part be explained by hampered CD4+ T cells effector functions in visceral leishmaniasis (VL) patients. In a recent studies that defined transcriptional signatures for CD4+ T cells from active VL patients, we found that expression of the IL-7 receptor alpha chain (IL-7Rα; CD127) was downregulated, compared to CD4+ T cells from endemic controls (ECs). Since IL-7 signaling is critical for the survival and homeostatic maintenance of CD4+ T cells, we investigated this signaling pathway in VL patients, relative to ECs. Methods: CD4+ T cells were enriched from peripheral blood collected from VL patients and EC subjects and expression of IL7 and IL7RA mRNA was measured by real time qPCR. IL-7 signaling potential and surface expression of CD127 and CD132 on CD4+ T cell was analyzed by multicolor flow cytometry. Plasma levels of soluble IL-7 and sIL-7Rα were measured by ELISA. Result: Transcriptional profiling data sets generated previously from our group showed lower IL7RA mRNA expression in VL CD4+ T cells as compared to EC. A significant reduction was, however not seen when assessing IL7RA mRNA by RT-qPCR. Yet, the levels of soluble IL-7Rα (sIL-7Rα) were reduced in plasma of VL patients compared to ECs. Furthermore, the levels of soluble IL-7 were higher in plasma from VL patients compared to ECs. Interestingly, expression of the IL-7Rα protein was higher on VL patient CD4+ T cells as compared to EC, with activated CD38+ CD4+ T cells showing higher surface expression of IL-7Rα compared to CD38- CD4+ T cells in VL patients. CD4+ T cells from VL patients had higher signaling potential baseline and after stimulation with recombinant human IL-7 (rhIL-7) compared to EC, as measured by phosphorylation of STAT5 (pSTAT5). Interestingly, it was the CD38 negative cells that had the highest level of pSTAT5 in VL patient CD4+ T cells after IL-7 stimulation. Thus, despite unaltered or potentially lowered IL7RA mRNA expression by CD4+ T cells from VL patients, the surface expression of the IL-7Rα was higher compared to EC and increased pSTAT5 was seen following exposure to rhIL-7. Accordingly, IL-7 signaling appears to be functional and even enhanced in VL CD4+ T cells and cannot explain the impaired effector function of VL CD4+ T cells. The enhanced plasma IL-7 may serve as part of homeostatic feedback mechanism regulating IL7RA expression in CD4+ T cells. Author summary: In visceral leishmaniasis (VL), antigen specific CD4+ T cell responses are muted hindering the control of the Leishmania donovani infection. IL-7 signaling is crucial for CD4+ T cell survival and function, and gene expression analysis indicated that the IL-7 pathway could be altered in VL. Thus, we investigate if impaired IL-7 signaling could explain the loss of antigen specific T cell response in VL. Although we didn't observe significant reduction of IL7RA mRNA by RT-qPCR, yet, the levels of soluble IL-7Rα (sIL-7Rα) were reduced in plasma of VL patients compared to ECs. Furthermore, the levels of soluble IL-7 were higher in plasma from VL patients compared to ECs and their CD4+ T cells exhibited heightened IL-7 receptor protein expression. Surprisingly, VL patient CD4+ T cells showed increased IL-7 signaling potential, as evidenced by higher phosphorylation of STAT5 upon IL-7 stimulation. While altered, the findings presented here do not attributed the impaired effector function of VL CD4+ T cells to defective IL-7 signaling. We speculate that the elevated plasma IL-7 is part of a homeostatic feedback mechanism in response to the reduced IL7RA transcription in CD4+ T cells. [ABSTRACT FROM AUTHOR]

The parasite Leishmania donovani is one of the species causing visceral leishmaniasis in humans, a deadly infection claiming up to 40,000 lives each year. The current drugs for leishmaniasis treatment have severe drawbacks and there is an urgent need to find new anti-leishmanial compounds. However, the search for drug candidates is complicated by the intracellular lifestyle of Leishmania. Here, we investigate the use of human induced pluripotent stem cell (iPS)-derived macrophages (iMACs) as host cells for L. donovani. iMACs obtained through embryoid body differentiation were infected with L. donovani promastigotes, and high-content imaging techniques were used to optimize the iMACs seeding density and multiplicity of infection, allowing us to reach infection rates up to 70% five days after infection. IC50 values obtained for miltefosine and amphotericin B using the infected iMACs or mouse peritoneal macrophages as host cells were comparable and in agreement with the literature, showing the potential of iMACs as an infection model for drug screening. Author summary: Author summary: Yearly, up two million people in poverty-stricken areas contract leishmaniasis, a disease caused by parasites of the genus Leishmania. When an infected sandfly takes a blood meal, Leishmania parasites enter the host where they are taken up by macrophages. Inside the macrophage, Leishmania parasites establish a niche where they can proliferate. The infection often leads to disability and can result in death. Moreover, the drugs currently available are unsatisfactory due to toxic side effects, high expenses or difficulties in usage. Drug screening assays that are currently used for compound screening often rely on mouse peritoneal macrophages. We have generated human induced pluripotent stem cell derived macrophages and used these as new host cells for Leishmania donovani in the testing of anti-leishmanial compounds. This model has many advantages. For one, it allows us to work with human cells, mimicking the natural infection more closely than murine cells. Secondly, it allows us to obtain bigger batches of uniform cells for screening campaigns. Finally, this approach aligns with the principles of the 3Rs, replacing the use of animals for cultivation of Leishmania and drug screening purposes. [ABSTRACT FROM AUTHOR]

Background: Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, a potentially fatal disease in humans and dogs. The epidemiology and transmission dynamics of L. donovani in Sudan are poorly understood. We aim to determine the prevalence and characterization of L. donovani in human hospital cases and domestic dogs using different diagnostic methods in two localities in Gadarif State, Sudan. Methods: A cross-sectional study was conducted from October 2019 to April 2020. Whole blood samples and lymph node aspirates were collected from 69 human and 32 dog participants for parasitological, immunological, and molecular tests. Results: No parasites were detected in blood samples from either humans or dogs. Lymph node microscopy revealed 71.01% positivity in humans and 9.4% in dogs. The IT Leish test showed 82.6% positivity in humans and 43.75% in dogs. PCR confirmed L. donovani infection in all six selected samples (three from humans and three from dogs). Conclusion: The study confirmed the presence of L. donovani in both human and dog populations in the study area, suggesting that dogs may act as reservoirs or hosts for the parasite. The IT Leish kit test was the most sensitive and specific method, while microscopy of blood and lymph node smears was the least sensitive method. Further studies are needed to elucidate the role of dogs in the transmission cycle of L. donovani and the risk factors associated with human infection. [ABSTRACT FROM AUTHOR]

Leishmania donovani, a protozoan parasite, resides and replicates in macrophages and inflicts the potentially fatal disease visceral leishmaniasis (VL). The parasite‐expressed surface lipophosphoglycan (LPG) was implicated in binding TLR2 on NK cells, but the modus operandi of its disease‐promoting influence remained unknown. As TPL2, a member of the MAPK module in mammalian macrophages, was implicated in the anti‐inflammatory immune response and promoting pathogen survival, we investigated the possibility of TPL2‐directed LPG‐TLR2 signalling in Leishmania infection. We observed that TLR2 or TPL2 blockade differentially influenced the TLR2 ligand proteoglycan (PGN)‐induced p38MAPK and ERK‐1/2 activation. TLR2 blockade abrogated the PGN‐induced TPL2 activation. L. donovani infection impaired the Akt activation whereas, upon TPL2 inhibition, the infection fails to control Akt phosphorylation. In L. donovani‐infected macrophages, TLR2 blocking negatively affected p38, Akt and TPL2 phosphorylation while ERK1/2 phosphorylation increased relative to the infection alone. TPL2 blockade reduced TGF‐β, but increased TNF‐α expression and diminished amastigote count in macrophages. While exploring stimulation patterns of TLR2 ligands, LPG, unlike PGN, selectively increased TLR2 expression in macrophages. LPG blockade increased p38MAPK and AKT, but slightly affected ERK‐1/2 and significantly reduced TPL2 phosphorylation from L. donovani‐infected macrophages. Molecular docking and molecular dynamics analysis drew a parallel between LPG's glycan chain lengths with the frequency of interaction with TLR2 which might impact TLR2 signalling. Therefore, the parasite regulates the TLR2 signalling via TPL2 when elicited by LPG‐TLR2 interaction for pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is a common neglected tropical disease in Ethiopia. Visceral leishmaniasis (VL) caused by Leishmania donovani presents in the lowlands, while cutaneous leishmaniasis (CL) affects people living in the highlands. Although CL is described as being caused by Leishmania aethiopica, there is also evidence of L. tropica and L. major isolated from a patient, sand flies and potential reservoirs. Information on species causing CL in Ethiopia is patchy, and no nation-wide study has ever been done. Understanding which species are causing CL in Ethiopia can have important implications for patient management and disease prevention. Methods: We analyzed stored routine samples and biobanked DNA isolates from previously conducted studies of CL patients from different centers in the north, center and south of Ethiopia. Species typing was performed using ITS-1 PCR with high-resolution melt (HRM) analysis, followed by HSP70 amplicon sequencing on a selection of the samples. Additionally, sociodemographic, clinical and laboratory data of patients were analyzed. Results: Of the 226 CL samples collected, the Leishmania species could be determined for 105 (45.5%). Leishmania aethiopica was identified in 101 (96.2%) samples from across the country. In four samples originating from Amhara region, northwestern Ethiopia, L. donovani was identified by ITS-1 HRM PCR, of which two were confirmed with HSP70 sequences. While none of these four patients had symptoms of VL, two originated from known VL endemic areas. Conclusions: The majority of CL was caused by L. aethiopica, but CL due to L. tropica and L. major cannot be ruled out. Our study is the first to our knowledge to demonstrate CL patients caused by L. donovani in Ethiopia. This should spark future research to investigate where, how and to which extent such transmission takes place, how it differs genetically from L. donovani causing VL and whether such patients can be diagnosed and treated successfully with the currently available tools and drugs. [ABSTRACT FROM AUTHOR]

A chromone-peptidyl hybrids series was synthesised and rationally repurposed towards identification of potential antileishmanial hits against visceral leishmaniasis. Three hybrids 7c, 7n, and 7h showed potential IC50 values (9.8, 10, and 12 µM, respectively) which were comparable to erufosine IC50 (9.8 µM) but lower potency than miltefosine IC50 (3.5 µM). Preliminary assessment of cytotoxicity using human THP-1 cells presented chromone-peptidyl hybrids 7c and 7n as non-cytotoxic up to 100 µM while erufosine and miltefosine had CC50 of 19.4 µM and >40 µM, respectively. In silico studies pinpointed the N-p-methoxyphenethyl substituent at the peptidyl moiety together with the oxygen-based substituted functions of the phenyl ring of the chromone moiety as crucial players in binding to LdCALP. Together, these findings present chromone-peptidyl hybrids 7c and 7n as potential and anticipated non-cytotoxic antileishmanial hit compounds for possible development of potential antileishmanial agents against visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL, kala azar), caused by Leishmania donovani, transmitted by Phlebotomus orientalis, is a serious systemic disease that causes high morbidity and mortality rates in Sudan and other parts of East Africa and the world. Despite progress in understanding the epidemiology of the disease in East Africa, little is known about the host preference of P. orientalis in kala azar endemic villages of Sudan, which have some of the highest VL incidence rates in the world. The present study used host choice experiments and blood‐meal identification approaches to determine the host preference of P. orientalis in kala azar endemic villages in Gedarif state, eastern Sudan. In the host choice experiment, tent traps were used to compare the attractiveness of cows, donkeys, sheep and goats for host‐seeking P. orientalis. In the blood‐meal identification study, blood‐fed P. orientalis females, captured inside houses and peri‐domestic habitats, were subjected to molecular typing using cytochrome b gene (cyt b) amplification and sequence analysis. Cows and donkeys were the most attractive to blood‐seeking P. orientalis, followed by goats. Similarly, the blood‐meal analysis of P. orientalis showed that the vector preferentially feeds on cows, followed by donkeys, humans and goats. The human blood index of P. orientalis was 19.4% (42/216), indicating a high zoophilic habit of the vector, both inside and outside the houses. Although the order of host preference varied by location, it was clear that cows are the most preferred host of P. orientalis in the area. Results are discussed in relation to the role of domestic/livestock animals in VL zoopotentiation and zooprophylaxis. Inference is made on the potential impact of insecticide treatment of cows in control of the vector and the transmission of VL in Sudan and other parts of East Africa. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL), a life-threatening neglected tropical disease, is targeted for elimination from Nepal by the year 2026. The national VL elimination program is still confronted with many challenges including the increasingly widespread distribution of the disease over the country, local resurgence and the questionable efficacy of the key vector control activities. In this study, we assessed the status and risk of Leishmania donovani transmission based on entomological indicators including seasonality, natural Leishmania infection rate and feeding behavior of vector sand flies, Phlebotomus argentipes, in three districts that had received disease control interventions in the past several years in the context of the disease elimination effort. Methods: We selected two epidemiologically contrasting settings in each survey district, one village with and one without reported VL cases in recent years. Adult sand flies were collected using CDC light traps and mouth aspirators in each village for 12 consecutive months from July 2017 to June 2018. Leishmania infection was assessed in gravid sand flies targeting the small-subunit ribosomal RNA gene of the parasite (SSU-rRNA) and further sequenced for species identification. A segment (~ 350 bp) of the vertebrate cytochrome b (cytb) gene was amplified from blood-fed P. argentipes from dwellings shared by both humans and cattle and sequenced to identify the preferred host. Results: Vector abundance varied among districts and village types and peaks were observed in June, July and September to November. The estimated Leishmania infection rate in vector sand flies was 2.2% (1.1%–3.7% at 95% credible interval) and 0.6% (0.2%–1.3% at 95% credible interval) in VL and non-VL villages respectively. The common source of blood meal was humans in both VL (52.7%) and non-VL (74.2%) villages followed by cattle. Conclusions: Our findings highlight the risk of ongoing L. donovani transmission not only in villages with VL cases but also in villages not reporting the presence of the disease over the past several years within the districts having disease elimination efforts, emphasize the remaining threats of VL re-emergence and inform the national program for critical evaluation of disease elimination strategies in Nepal. [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]

A series of rosmarinic acid-β-amino-α-ketoamide hybrids were synthesized and rationally repurposed towards the identification of new antileishmanial hit compounds. Two hybrids, 2g and 2h, showed promising activity (IC50 values of 9.5 and 8.8 μM against Leishmania donovani promastigotes, respectively). Their activities were comparable to erufosine. In addition, cytotoxicity evaluation employing human THP-1 cells revealed that the two hybrids 2g and 2h possess no cytotoxic effects up to 100 µM, while erufosine possessed cytotoxicity with CC50 value of 19.4 µM. In silico docking provided insights into structure–activity relationship emphasizing the importance of the aliphatic chain at the α-carbon of the cinnamoyl carbonyl group establishing favorable binding interactions with LdCALP and LARG in both hybrids 2g and 2h. In light of these findings, hybrids 2g and 2h are suggested as potential safe antileishmanial hit compounds for further development of anti-leishmanial agents. [ABSTRACT FROM AUTHOR]

We sequenced Leishmania donovani genomes in blood samples collected in emerging foci of visceral leishmaniasis in western Nepal. We detected lineages very different from the preelimination main parasite population, including a new lineage and a rare one previously reported in eastern Nepal. Our findings underscore the need for genomic surveillance. [ABSTRACT FROM AUTHOR]

Leishmaniasis, a neglected tropical disease, encompasses a spectrum of clinical conditions and poses a significant risk of infection to over one billion people worldwide. Visceral leishmaniasis (VL) in the Indian sub-continent (ISC), where the causative parasite is Leishmania donovani, is targeted for elimination by 2025, with some countries already reaching such targets. Other clinical phenotypes due to the same species could act as a reservoir of parasites and thus pose a challenge to successful control and elimination. Sri Lanka has consistently reported cutaneous leishmaniasis (CL) due to L. donovani as the primary disease presentation over several decades. Similar findings of atypical phenotypes of L. donovani have also been reported from several other countries/regions in the Old World. In this review, we discuss the applicability of different methods in diagnosing CL due to L. donovani and a comprehensive assessment of diagnostic methods spanning clinical, microscopic, molecular, and immunological approaches. By incorporating evidence from Sri Lanka and other regions on L. donovani-related CL, we thoroughly evaluate the accuracy, feasibility, and relevance of these diagnostic tools. We also discuss the challenges and complexities linked to diagnosing CL and review novel approaches and their applicability for detecting CL. [ABSTRACT FROM AUTHOR]

Leishmania donovani causes the neglected tropical disease visceral leishmaniasis. There is currently no vaccine available to combat the disease. Therefore, chemotherapy is the only treatment option available. However, the available drugs have various adverse effects on the host. As a result, new drugs are needed to treat the disease; natural compounds can be used as an alternative to traditional drugs. This study examined apigenin, diosmin, and naringin as prospective inhibitors against the Leishmania donovani Sterol 24-C- methyltransferase enzyme (LdSMT). All three compounds have a high affinity for the LdSMT and remain stable for 100 ns during the molecular dynamics simulation. In the cell viability assays, these compounds showed excellent IC50 values, with apigenin having the best IC50 value of 16.11 µM against Leishmania donovani promastigote. The findings of this study showed that all three flavonoids could be used as a safe alternative to conventional anti-leishmanial drugs. [ABSTRACT FROM AUTHOR]

Simple Summary: Different diagnoses for Feline leishmaniasis have been studied, mainly in endemic regions of Canine Visceral Leishmaniasis (CVL), aiming to contribute to the differentiation of diseases manifested by cats with the aim of offering the best treatment for the animal. However, the study of less invasive and sensitive diagnostic techniques can be useful concomitantly with serological techniques such as the Immunofluorescent Antibody Test (IFAT) and ELISA in epidemiological surveys. In this sense, the objective of our study was to evaluate positivity for Leishmania from material collected in a less invasive way in serologically positive and negative cats from an endemic region for CVL; through conjunctival swabs for molecular analysis by PCR, with subsequent screening of the species by the HRM technique; and confirmation by genetic sequencing. Although few samples could be detected from the conjunctival cell samples (4/36), there was a good concordance of species confirmation by the HRM technique and genetic sequencing. Of these four animals, three were confirmed as positive by the two serological techniques, which suggests that they had contact with the parasite and produced antibodies. Therefore, HRM would be an alternative as a screening technique for different species of Leishmania followed by confirmation by genetic sequencing. Background and objectives: Feline leishmaniasis (FeL) is caused by several species of parasites of the genus Leishmania. The disease can occur with the presence or absence of clinical signs, similar to those observed in other common infectious diseases. In endemic regions for FeL, the infection has been associated with dermatological lesions. Therefore, considering the search for less invasive and more effective diagnostic techniques, we aimed to investigate the presence of Leishmania spp. in domestic cats through Polymerase Chain Reaction (PCR) and high-resolution melting (HRM) analyses of conjunctival, oral, and nasal epithelial cells, and we detected the presence of anti-Leishmania IgG antibodies from serological techniques of the Immunofluorescent Antibody Test (IFAT) and ELISA. Methods: The PCR and HRM for detection of Leishmania spp. were performed on 36 samples of epithelial cells from the conjunctiva of male and female cats, collected using sterile swabs. The serological tests IFAT and ELISA were also performed. Results: The prevalence of Leishmania donovani infection was 11.1% (4/36) by PCR assay, and those results were confirmed for Leishmania species using the HRM technique. Twenty-four cats (24/36 = 66.7%) were reactive to the IFAT and twenty-two cats were reactive by the ELISA technique (22/36 = 61.1%). Interpretation and Conclusions: The use of conjunctival swabs was shown to be a non-invasive, practical, and easy-to-perform technique, and in addition to the genetic sequencing and HRM, it was able to identify the parasitic DNA of L. donovani in cats. This technique can be used for screening diagnosis in future epidemiological surveys of FeL and can be used as a complement to clinical and/or serological tests, as well as associating the clinical history of the animal, for the diagnostic conclusion. [ABSTRACT FROM AUTHOR]

Leishmania donovani is the causative organism for visceral leishmaniasis. Although this parasite was discovered over a century ago, nothing is known about role of potassium channels in L. donovani. Potassium channels are known for their crucial roles in cellular functions in other organisms. Recently the presence of a calcium-activated potassium channel in L. donovani was reported which prompted us to look for other proteins which could be potassium channels and to investigate their possible physiological roles. Twenty sequences were identified in L. donovani genome and subjected to estimation of physio-chemical properties, motif analysis, localization prediction and transmembrane domain analysis. Structural predictions were also done. The channels were majorly α-helical and predominantly localized in cell membrane and lysosomes. The signature selectivity filter of potassium channel was present in all the sequences. In addition to the conventional potassium channel activity, they were associated with gene ontology terms for mitotic cell cycle, cell death, modulation by virus of host process, cell motility etc. The entire study indicates the presence of potassium channel families in L. donovani which may have involvement in several cellular pathways. Further investigations on these putative potassium channels are needed to elucidate their roles in Leishmania. [ABSTRACT FROM AUTHOR]

Eukaryotic cells have distinct membrane-enclosed organelles, each with a unique biochemical signature and specialized function. The unique identity of each organelle is greatly governed by the asymmetric distribution and regulated intracellular movement of two important biomolecules, lipids, and proteins. Non-vesicular lipid transport mediated by lipid-transfer proteins (LTPs) plays essential roles in intra-cellular lipid trafficking and cellular lipid homeostasis, while vesicular transport regulates protein trafficking. A comparative analysis of non-vesicular lipid transport machinery in protists could enhance our understanding of parasitism and basis of eukaryotic evolution. Leishmania donovani, the trypanosomatid parasite, greatly depends on receptor-ligand mediated signalling pathways for cellular differentiation, nutrient uptake, secretion of virulence factors, and pathogenesis. Lipids, despite being important signalling molecules, have intracellular transport mechanisms that are largely unexplored in L. donovani. We have identified a repertoire of sixteen (16) potential lipid transfer protein (LTP) homologs based on a domain-based search on TriTrypDB coupled with bioinformatics analyses, which signifies the presence of well-organized lipid transport machinery in this parasite. We emphasized here their evolutionary uniqueness and conservation and discussed their potential implications for parasite biology with regards to future therapeutic targets against visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is caused by Leishmania donovani (Ld), and most cases occur in Brazil, East Africa, and India. The treatment for VL is limited and has many adverse effects. The development of safer and more efficacious drugs is urgently needed. Drug repurposing is one of the best processes to repurpose existing drugs. Ornithine decarboxylase (ODC) is an important target against L. donovani in the polyamine biosynthesis pathway. In this study, we have modeled the 3D structure of ODC and performed high‐throughput virtual screening of 8630 ZINC database ligands against Leishmania donovani ornithine decarboxylase (Ld ODC), selecting 45 ligands based on their high binding score. It is further validated through molecular docking simulation and the selection of the top two lead molecules (ceftaroline fosamil and rimegepant) for Molecular Dynamics (MD) simulation, Density functional theory (DFT), and molecular mechanics generalized born surface area (MMGBSA) analysis. The results showed that the binding affinities of ceftaroline fosamil, and rimegepant are, respectively, −10.719 and 10.159 kcal/mol. The docking complexes of the two lead compounds, ceftaroline fosamil, and rimegepant, with the target ODC, were found stable during molecular dynamics simulations. Furthermore, the analysis of MMGBSA revealed that these compounds had a high binding free energy. The DFT analysis showed that the top lead molecules were more reactive than the standard drug (pentamidine). In‐silico findings demonstrated that ceftaroline fosamil, and rimegepant might be recognized as potent antagonists against ODC for the treatment of VL. [ABSTRACT FROM AUTHOR]

Drug repurposing has emerged as an effective strategy against infectious diseases such as visceral leishmaniasis. Here, we evaluated four FDA‐approved drugs–valrubicin, ciclesonide, deflazacort, and telithromycin—for their anti‐leishmanial activity on Leishmania donovani parasites, especially their ability to target the enzyme glutathione synthetase (LdGS), which enables parasite survival under oxidative stress in host macrophages. Valrubicin and ciclesonide exhibited superior inhibitory effects compared to deflazacort and telithromycin, inhibiting the promastigotes at very low concentrations, with IC50 values of 1.09 ± 0.09 μm and 2.09 ± 0.09 μm, respectively. Subsequent testing on amastigotes revealed the IC50 values of 1.74 ± 0.05 μm and 3.32 ± 0.21 μm for valrubicin and ciclesonide, respectively. Molecular and cellular level analysis further elucidated the mechanisms underlying the anti‐leishmanial activity of valrubicin and ciclesonide. [ABSTRACT FROM AUTHOR]

Cutaneous leishmaniasis caused by Leishmania major or L. tropica and visceral leishmaniasis caused by L. infantum have been reported in Israel. We collected Phlebotomus spp. sand flies in the Negev desert of southern Israel to identify circulating Leishmania spp. Of 22,636 trapped sand flies, 80% were P. alexandri. We sequenced Leishmania-specific internal transcribed spacer 1 fragments and K26 genes. Of 5,019 Phlebotomus female sand flies, 2.5% were Leishmania DNA–positive; 92% of infections were L. donovani. Phylogenetic analyses showed separate clustering of L. donovani and L. infantum. P. alexandri flies positive for L. donovani harbored blood meals from European hares. Leishmania DNA isolated from a patient with cutaneous leishmaniasis who lived in the survey area was identical to L. donovani from P. alexandri flies. We report circulation of L. donovani, a cause of visceral leishmaniasis, in southern Israel. Prompt diagnosis and Leishmania spp. identification are critical to prevent leishmaniasis progression. [ABSTRACT FROM AUTHOR]

Background: The potential reservoirs of visceral leishmaniasis (VL) in South Asia include asymptomatic and relapsed cases of VL, along with patients with post kala-azar dermal leishmaniasis (PKDL). Accordingly, accurate estimation of their parasite load is pivotal for ensuring disease elimination, presently targeted for 2023. Serological tests cannot accurately detect relapses and/or monitor treatment effectiveness, and therefore, parasite antigen/nucleic acid based detection assays remain the only viable option. An excellent option is the quantitative polymerase chain reaction (qPCR) but the high cost, technical expertise and time involved precludes its wider acceptability. Accordingly, the recombinase polymerase amplification (RPA) assay operated in a mobile suitcase laboratory has emerged not simply as a diagnostic tool for leishmaniasis but also to monitor the disease burden. Methodology/Principal findings: Using total genomic DNA isolated from peripheral blood of confirmed VL cases (n = 40) and lesional biopsies of PKDL cases (n = 64), the kinetoplast-DNA based qPCR and RPA assay was performed and parasite load expressed as Cycle threshold (Ct) and Time threshold (Tt) respectively. Using qPCR as the gold standard, the diagnostic specificity and sensitivity of RPA in naïve cases of VL and PKDL was reiterated. To assess the prognostic potential of the RPA, samples were analyzed immediately at the end of treatment or ≥6 months following completion of treatment. In cases of VL, the RPA assay in terms of cure and detection of a relapse case showed 100% concordance with qPCR. In PKDL following completion of treatment, the overall detection concordance between RPA and qPCR was 92.7% (38/41). At the end of treatment for PKDL, 7 cases remained qPCR positive, whereas RPA was positive in only 4/7 cases, perhaps attributable to their low parasite load. Conclusions/Significance: This study endorsed the potential of RPA to evolve as a field applicable, molecular tool for monitoring parasite load, possibly at a point of care level and is worthy of consideration in resource limited settings. Author summary: Visceral leishmaniasis (VL) or kala-azar caused by the digenetic parasite Leishmania donovani is a fatal form of leishmaniasis, unless treated. In South Asia, approximately 10–20% of apparently cured cases of VL develop a dermal sequela termed post kala-azar dermal leishmaniasis (PKDL). It presents with macular or papulonodular lesions that harbour Leishmania parasites. Importantly, these cases of PKDL in addition to asymptomatic and relapsed VL cases serve as mobile disease reservoirs and sustain disease transmission. In macular PKDL, that presently comprise over 50% of cases, the diagnosis is particularly challenging as parasites are often not detectable by microscopy. Accordingly, development of strategies for molecular diagnosis is one of the major foci of the ongoing leishmaniasis elimination programme targeted for 2023, that aims to decrease the burden to <1/10,000 cases of VL at the block/zilla level. Serological tests have limited applicability when it is necessary to distinguish present from past infection of VL, and in cases of PKDL when the positivity maybe due to a past history of VL. An excellent option is the quantitative polymerase chain reaction (qPCR) but limitations for its wider field applicability include the high cost, technical expertise and time factor. Accordingly, an urgent need exists for availability of field applicable molecular tests. The RPA tested in this study could well be the desired game-changer as it provided accurate diagnosis of VL and PKDL, can be performed in a primary health centre, and demonstrated the potential to be a monitoring tool for parasite load after treatment, that eventually can accelerate achieving the end-goal of elimination of leishmaniasis. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is a neglected tropical disease that is globally distributed and has the potential to cause very serious illness. Prior literature highlights the emergence and spread of VL is influenced by multiple factors, such as socioeconomic status, sanitation levels or animal and human reservoirs. The study aimed to retrospectively investigate the presence and infectiousness of VL in Rio Grande do Norte (RN), Brazil between 2007 and 2020. We applied a hierarchical Bayesian approach to estimate municipality-specific relative risk of VL across space and time. The results show evidence that lower socioeconomic status is connected to higher municipality-specific VL risk. Overall, estimates reveal spatially heterogeneous VL risks in RN, with a high probability that VL risk for municipalities within the West Potiguar mesoregion are more than double the expected VL risk. Additionally, given the data available, results indicate there is a high probability of increasing VL risk in the municipalities of Natal, Patu and Pau dos Ferros. These findings demonstrate opportunities for municipality-specific public health policy interventions and warrant future research on identifying epidemiological drivers in at-risk regions. Author summary: As a neglected tropical disease (NTD), Leishmaniasis contributes to poor health outcomes, suffering, and death of vulnerable populations. Leishmania infantum or Leishmania donovani is transmitted from animals to humans through the sandfly vector and can be fatal in 5% of the cases even with treatment. Visceral leishmaniasis is a severe form of Leishmaniasis. The purpose of this study was to estimate regions of high Visceral leishmaniasis risk in Rio Grande do Norte, Brazil between 2007 and 2020 to help inform public health initiatives and potential health policy interventions for this disease. Visceral leishmaniasis is a reportable disease and therefore VL cases are documented to the Brazilian Ministry of Health database through passive surveillance. Passive surveillance data can suffer from missing information; individuals may be asymptomatic, or may not seek medical care allowing for diagnosis. When modeling disease risk from passive surveillance data, assuming there is an absence of disease in regions without documented cases can underestimate disease risk. In this paper we estimate disease risk and identify areas with high-risk and increasing infection trends over time while considering data characteristics such as missing information and high correlation of risk factors. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is a neglected disease with a broad spectrum of clinical manifestations and involvement of visceral organs. Organ‐specific immune response against the Leishmania donovani (Ld) complex is not yet understood due to the unavailability of an appropriate experimental model. In reference to our recent work on comparing the hamster model with VL patients, it is now possible to understand immune profiling in different visceral organs. This may offer an answer to varying parasite loads in different visceral organs in the same host. Herein, we analysed a panel of immune markers (Th‐2/Th‐1) in visceral organs of Ld‐infected hamsters and quantified parasitic load in the same tissues using qPCR assay. In spleen, liver, bone marrow and lymph node (mesenteric) from Ld‐infected hamsters, the parasite burden was quantified along with mRNA expression of a panel of Th‐2 and Th‐1 type immune markers, namely IL‐10, IL‐4, Arginase‐I, GATA‐3, SOCS‐3, IL‐12, IFN‐γ, iNOS, T‐bet and SOCS‐5. A clear dichotomy was absent between Th‐2 and Th‐1 type immune markers and the major players of this immune response were IFN‐γ, IL‐10, T‐bet, GATA‐3, SOCS‐5 and SOCS‐3. [ABSTRACT FROM AUTHOR]

Background: As untreated visceral leishmaniasis (VL) is fatal, reliable diagnostics are pivotal for accurate treatment allocation. The current diagnostic algorithm for VL in Ethiopia, which is based on the rK39 rapid diagnostic test and microscopy of tissue smears, lacks sensitivity. This probably leads to missed cases and patients not receiving treatment. Methodology: We conducted a retrospective study on stored microscopically negative spleen and bone marrow smears from suspected VL patients collected at the Leishmaniasis Research and Treatment Center (LRTC) in Gondar, northern Ethiopia between June 2019 and November 2020. Sociodemographic, clinical and treatment data were collected and samples were tested by real-time PCR targeting kinetoplast DNA. Principle findings: Among the 191 eligible samples (135 spleen and 56 bone marrow) with a microscopically negative and valid PCR result, 119 (62.3%) were positive by PCR, although Ct values for some were high (median 33.0). Approximately three quarters of these undiagnosed primary VL (77.3%) and relapse (69.6%) patients did not receive antileishmanial treatment. Of the 56 microscopically negative bone marrow samples, 46 (82.1%) were PCR positive, which is considerably higher compared to the microscopically negative spleen samples, for which 73 out of 135 (54.1%) were PCR positive. The odds of being PCR positive were significantly higher for bone marrow aspirates and higher when white blood cell values were lower and splenomegaly (in cm) was more pronounced. Conclusions: This study demonstrates that a lot of suspected VL patients remain undiagnosed and untreated. This indicates the urgent need for better diagnostics for VL in the East-African region. The outcomes of PCR positive should be closely monitored and treatment should be provided if the patient deteriorates. In resource limited settings, implementation of PCR on bone marrow aspirate smears of patients with low WBC values and splenomegaly could lead to considerable improvements in patient management. Author summary: As untreated visceral leishmaniasis (VL) is fatal, reliable diagnostics are important for accurate treatment allocation. The current diagnostic algorithm for VL in Ethiopia, which is based on the rK39 rapid diagnostic test and microscopy of tissue smears, lacks sensitivity. This probably leads to missed cases and patients not receiving treatment. To prove this, we conducted a study on stored microscopically negative spleen and bone marrow aspirate smears from suspected VL patients in Gondar, Ethiopia. Clinical and treatment data were collected and samples were tested for Leishmania by PCR. We found that about 60% of these microscopically negative samples were PCR positive. This PCR positivity rate was considerably higher in patients with a microscopically negative bone marrow compared to splenic aspirate. Importantly, more than three quarters of the patients with a PCR positive sample was not treated for VL. Overall, our study demonstrates the gap in the diagnostic algorithm for VL in northern Ethiopia, especially when bone marrow samples are used. In resource limited settings, we advise to challenge the current diagnostic algorithm and implement molecular tools to accurately diagnose patients. This could lead to considerable improvements in patient management in Ethiopia and beyond. [ABSTRACT FROM AUTHOR]

Background: In Nepal, the burden of post kala-azar dermal leishmaniasis (PKDL) is not known since there is no active case detection of PKDL by the national programme. PKDL patients could pose a challenge to sustain visceral leishmaniasis (VL) elimination. The objective of this study was to determine the prevalence of PKDL and assess PKDL patients' knowledge on VL and PKDL, and stigma associated with PKDL. Methodology/Principal findings: Household surveys were conducted in 98 VL endemic villages of five districts that reported the highest number of VL cases within 2018–2021. A total of 6,821 households with 40373 individuals were screened for PKDL. Cases with skin lesions were referred to hospitals and examined by dermatologists. Suspected PKDL cases were tested with rK39 and smear microscopy from skin lesions. An integrated diagnostic approach was implemented in two hospitals with a focus on management of leprosy cases where cases with non-leprosy skin lesions were tested for PKDL with rK39. Confirmed PKDL patients were interviewed to assess knowledge and stigma associated with PKDL, using explanatory model interview catalogue (EMIC) with maximum score of 36. Among 147 cases with skin lesions in the survey, 9 (6.12%) were confirmed as PKDL by dermatologists at the hospital. The prevalence of PKDL was 2.23 per 10,000 population. Among these 9 PKDL cases, 5 had a past history of VL and 4 did not. PKDL cases without a past history of VL were detected among the "new foci", Surkhet but none in Palpa. None of the cases negative for leprosy were positive for PKDL. There was very limited knowledge of PKDL and VL among PKDL cases. PKDL patients suffered to some degree from social and psychological stigma (mean ± s.d. score = 17.89 ± 12.84). Conclusions/Significance: Strengthening the programme in PKDL case detection and management would probably contribute to sustenance of VL elimination. Awareness raising activities to promote knowledge and reduce social stigma should be conducted in VL endemic areas. Author summary: Post kala-azar dermal leishmaniasis (PKDL) is a sequel of visceral leishmaniasis (VL) and occurs in VL endemic areas. PKDL patients can be a reservoir for Leishmania donovani parasites and thus sustaining the occurrence of VL. Since Bangladesh, India and Nepal have targeted the elimination of VL, it is important to eliminate PKDL as well. However, in Nepal there is no active case detection of PKDL and the real burden of PKDL is not known. This community based survey encountered a PKDL prevalence of 2.23 per 10,000 population. PKDL patients had limited knowledge of VL and PKDL. They suffered to some degree from social and psychological stigma. Therefore, PKDL case detection and management should be strengthened in the national programme in the context of the VL elimination initiative. [ABSTRACT FROM AUTHOR]

Objectives Previously, a series of side chain-modified quinolinyl β-enaminones was identified to possess significant activity against chloroquine-sensitive or -resistant Plasmodium falciparum and Brugia malayi microfilariae. The present study evaluates in vitro and in vivo activity of the series against Leishmania donovani and reports their mode of action. Methods The in vitro activity of 15 quinolinyl β-enaminone derivatives against Leishmania promastigotes and amastigotes was assessed by luciferase assay. The reduction of organ parasite burden was assessed by Giemsa staining in L. donovani -infected BALB/c mice and hamsters. Intracellular Ca2+ and ATP level in active derivative (3D)-treated promastigotes were determined by fluorescence and luminescence assays. Flow cytometry was performed to determine loss of mitochondrial membrane potential (MMP) using JC-1 dye, reactive oxygen species (ROS) generation using 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) dye, phosphatidylserine externalization by Annexin V-FITC staining and cell-cycle arrest by propidium iodide (PI) staining. Results Compounds 3A, 3B and 3D showed significant in vitro efficacy against L. donovani with IC50 < 6 µM and mild cytotoxicity (∼75% viability) at 25 µM on J774 macrophages. 3A and 3D at 50 mg/kg and 100 mg/kg reduced parasite burden (>84%) in infected mice and hamsters, respectively, whereas 3D-treated animals demonstrated maximum parasite burden reduction without organ toxicity. Mode-of-action analysis revealed that 3D induced apoptosis by inhibiting mitochondrial complex II, reducing MMP and ATP levels, increasing ROS and Ca2+ levels, ultimately triggering phosphatidylserine externalization and sub-G0/G1 cell-cycle arrest in promastigotes. Conclusions Compound 3D-mediated inhibition of L. donovani mitochondrial complex induces apoptosis, making it a promising therapeutic candidate for visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL), the second-most-serious parasitic illness after malaria, is currently endemic in more than 88 countries. Need for new antileishmanial compounds is currently being taken into consideration by researchers due to resistance and lack of effective vaccinations. This research was conducted to find out more about the effect of artemisinin (ART). ART was examined in vitro promastigotes stages and ex vivo amastigotes stages of the Iraqi strain of Leishmania donovani in U937 cell line after 24, 48 and 27 hours using MTT assay. In addition, the level of macrophage nitric oxide (NO) was measured using Griess assay in U937 cell line. The results of promastigotes viability percentage were less than 50 %, where the IC50 was 43.8, 37.9 and 36.6 µM respectively. Similar cytotoxic effect of ART was observed against amastigote forms, where the cell viability was 36.11, 40.2 and 37.4 µM respectively, after the three times of follow up. Furthermore, by using Griess assay the level of nitrite in infected macrophages was measured. The results after 48hr were 6.58 in high concentration and 1.22 in lower concentration. However, considering the absence of a significant difference, the observed concentrations are still higher than the control when compared to Griess assay. These findings demonstrated that ART has a cytotoxic effect on both forms of L. donovani. It also increased iNOS production in infected macrophages which can define artemisinin therapeutic efficacy against L. donovani infection. [ABSTRACT FROM AUTHOR]

Parasite of genus Leishmania viz. L. donovani and L. infantum cause visceral leishmaniasis (VL) or Kala-azar, systemic disease with significant enlargement of the liver and spleen, weight loss, anemia, fever and immunosuppression. The silent expansion of vectors, reservoir hosts and resistant strains is also of great concern in VL control. Considering all these issues, the present study focused on in vitro and in vivo antileishmanial screening of ellagic acid (EA) against L. donovani. The in vitro study was performed against the protozoan parasite L. donovani and a 50% inhibitory concentration was calculated. The DNA arrest in the sub-G0/G1 phase of the cell cycle was studied. In vivo studies included the assessment of parasite burden and immunomodulation in response to treatment of ellagic acid in BALB/c mice. The levels of Th1 and Th2 cytokines and isotype antibodies were assessed in different groups of mice. EA showed in vitro parasiticidal activity with IC50 18.55 µg/mL and thwarted cell-cycle progression at the sub-G0/G1 phase. Administration of ellagic acid to the BALB/c mice reported diminution of splenic and hepatic parasite burden coupled with an expansion of CD4+ and CD8+ T lymphocytes. EA further potentiated a protective immune response with augmentation of Th1 type immune response evidenced by elevation of serum IgG2a levels and DTH response. EA was reported to be safe and non-toxic to the THP-1 cell line as well as to the liver and kidneys of mice. These findings endorse the therapeutic potential of EA with significant immunomodulation and can serve as a promising agent against this debilitating parasitic disease. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis is a neglected vector-borne tropical disease caused by Leishmania donovani and Leishmania infantum that is endemic not only in East African countries, but also in Asia, regions of South America and the Mediterranean Basin. For the pharmacological control of this disease, there is a limited number of old and, in general, poorly adherent drugs, with a multitude of adverse effects and low oral bioavailability, which favor the emergence of resistant pathogens. Pentavalent antimonials are the first-line drugs, but due to their misuse, resistant Leishmania strains have emerged worldwide. Although these drugs have saved many lives, it is recommended to reduce their use as much as possible and replace them with novel and more friendly drugs. From a commercial collection of anti-infective drugs, we have recently identified nifuratel—a nitrofurantoin used against vaginal infections—as a promising repurposing drug against a mouse model of visceral leishmaniasis. In the present work, we have tested combinations of miltefosine—the only oral drug currently used against leishmaniasis—with nifuratel in different proportions, both in axenic amastigotes from bone marrow and in intracellular amastigotes from infected Balb/c mouse spleen macrophages, finding a potent synergy in both cases. In vivo evaluation of oral miltefosine/nifuratel combinations using a bioimaging platform has revealed the potential of these combinations for the treatment of this disease. [ABSTRACT FROM AUTHOR]

Background: The innate immune mediators are likely to influence the clinical phenotype of leishmaniasis by primary responses which limit or facilitate the spread of the parasite, as well as by modulating adaptive immunity. This study investigated the response of key innate immune cells in a focus which regularly reports localised cutaneous leishmaniasis (LCL) caused by Leishmania donovani, a species which typically causes visceral disease. Methods: Peripheral blood mononuclear cell (PBMC) derived macrophages and dendritic cells from patients with LCL and healthy controls from endemic and non-endemic areas, were stimulated with soluble Leishmania antigen (SLA). Inflammatory mediators produced by macrophages (TNF-α/TGF-β/IL-10, ELISA; NO, Griess method) and dendritic cells (IL-12p70, IL-10, flowcytometry) and macrophage expression of surface markers of polarization, activation and maturation (flowcytometry) were determined at 24h, 48h and 72h and compared. Study was conducted prospectively from 2015–2019. Results: Patient derived macrophages and dendritic cells produced higher levels of both pro and anti-inflammatory mediators compared to controls (p<0.05) with the best discrimination for active disease observed at 72h. Data demonstrated an early activation of macrophages and a subsequent pro-inflammatory bias, as indicated by temporal profiles of TNF-α/TGF-β and TNF-α/IL-10 ratios and higher proportions of classical (M1) macrophages. Higher TGF-β levels were observed in cells from patients with ulcerated or persistent lesions. Immune responses by cells derived from controls in endemic and non-endemic regions did not differ significantly from each other. Conclusions: The overall immunophenotypic profile suggests that LCL observed in the country is the result of a balancing immune response between pro-inflammatory and regulatory mediators. The mediators which showed distinct profiles in patients warrant further investigation as potential candidates for immunotherapeutic approaches. A comparison with visceral leishmaniasis caused by the same species, would provide further evidence on the differential role of these mediators in the resulting clinical phenotype. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis is caused by the Leishmania donovani species complex that can spread to internal organs and leading to death if not treated on time. Diagnosis of leishmaniasis is based on clinical signs and symptoms, microscopy, serological and molecular techniques. Because of a broad spectrum of diverse clinical manifestations and similarities of the responses to different species, identification to the species level is often difficult for the proper patient treatment and management. Therefore, the objective of this study was to evaluate the PCR- RFLP assay of the ITS1 region for identification of L. donovani species from clinical smear slide patient samples. Method: DNA extraction was performed on a total of 90 smear slide samples using phenol—chloroform method. The PCR detection limit was determined by L. donovani reference strain DNA. The ITS1 region was amplified at 320 bp using LITSR/L5.8S genus specific primers and then the ITS1-PCR products were subjected to RFLP assay for confirmation of L. donovani species using HaeIII restriction enzyme. Results: Of the total samples ITS1-PCR revealed the true positive, false positive, true negative, and false negative results of 42 (46.7%), 6 (6.7%), 37 (41.1%) and 5 (5.6%), respectively. Considering microscopy as the gold standard, the sensitivity, specificity, positive predictive values, and negative predictive values of the ITS1- PCR technique was 89.4%, 86.0%, 87.5%, and 88.1% respectively. All ITS1-PCR positive clinical samples were confirmed as L. donovani species by PCR–RFLP patterns. Conclusion: In conclusion, the ITS1- RFLP method is highly sensitive and more specific for identification of L. donovani species in the smear negative clinical samples of visceral leishmaniasis patients. There is also significant association and degree of agreement between the two methods. For direct identification of L. donovani species from clinical samples, irrespective of genus and species level, PCR–RFLP is more recommendable than a microscope. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) is the most severe form of all leishmanial infections and is caused by infection with protozoa of Leishmania donovani and Leishmania infantum. This parasitic disease occurs in over 80 countries and its geographic distribution is on the rise. Although the interaction between the intestinal microbiome and the immune response has been established in several pathologies, it has not been widely studied in leishmaniasis. The Syrian hamster is the most advanced laboratory model for developing vaccines and new drugs against VL. In the study reported here, we explored the relationship between the intestinal microbiome and infection with L. infantum in this surrogate host. Methods: Male Syrian hamsters (120–140 g) were inoculated with 108 promastigotes of a canine-derived L. infantum strain or left as uninfected control animals. Infection was maintained for 19 weeks (endpoint) and monitored by an immunoglobulin G (IgG) enyzme-linked immunosorbent assay throughout the experiment. Individual faecal samples, obtained at weeks 16, 18 and 19 post-inoculation, were analysed to determine the 16S metagenomic composition (the operational taxonomic units [OTUs] of the intestinal microbiome and the comparison between groups were FDR (false discovery rate)-adjusted). Results: Leishmania infantum infection elicited moderate clinical signs and lesions and a steady increase in specific anti-Leishmania serum IgG. The predominant phyla (Firmicutes + Bacteriodetes: > 90%), families (Muribaculaceae + Lachnospiraceae + Ruminococcaceae: 70–80%) and genera found in the uninfected hamsters showed no significant variations throughout the experiment. Leishmania infantum infection provoked a slightly higher—albeit non-significant—value for the Firmicutes/Bacteriodetes ratio but no notable differences were found in the relative abundance or diversity of phyla and families. The microbiome of the infected hamsters was enriched in CAG-352, whereas Lachnospiraceae UCG-004, the [Eubacterium] ventriosum group and Allobaculum were less abundant. Conclusions: The lack of extensive significant differences between hamsters infected and uninfected with L. infantum in the higher taxa (phyla, families) and the scarce variation found, which was restricted to genera with a low relative abundance, suggest that there is no clear VL infection-intestinal microbiome axis in hamsters. Further studies are needed (chronic infections, co-abundance analyses, intestinal sampling, functional analysis) to confirm these findings and to determine more precisely the possible relationship between microbiome composition and VL infection. [ABSTRACT FROM AUTHOR]

Leishmania donovani and Leishmania infantum are closely related species. However, the former is considered the causative agent for anthroponotic visceral leishmaniasis (AVL), while the latter is known to be responsible for zoonotic visceral leishmaniasis (ZVL) with dogs as the main reservoir host. Although molecular detection of L. donovani from naturally infected dogs has been reported in AVL endemic areas, the experimental infection of dogs with this species is very limited. Here, we constructed an experimental canine visceral leishmaniasis (CVL) model with L. donovani infection using beagle dogs. During an observation period of 8 months after parasite inoculation, few clinical symptoms were observed in the three inoculated dogs. The overall hematological and biochemical data of the dogs showed normal levels, and there were no remarkable changes in the peripheral CD4+, CD8+, CD25+, or FoxP3+ T cell populations. Liver biopsy sampling was conducted to monitor the parasite burden in the liver. A similar pattern of the amount of mitochondrial kinetoplast DNA was observed in the peripheral blood and liver by real-time PCR analysis. In addition, parasite antigens were detected from the liver biopsy sections by immunohistochemical analysis, further supporting the existence of parasites in the liver. These results showed a subclinical CVL model for L. donovani in beagle dogs with a similar kinetics of parasite burden in the peripheral blood and liver. [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]

In the absence of adequate diagnosis and treatment, leishmaniasis remains a major public health concern on a global scale. Drug resistance remains a key obstacle in controlling and eliminating visceral leishmaniasis. The therapeutic gap due to lack of target-specific medicine and vaccine can be minimized by obtaining parasite's genomic information. This study compared whole-genome sequence of paromomycin-resistant parasite (K133PMM) developed through in vitro adaptation and selection with sensitive Leishmania clinical isolate (K133WT). We found a large number of upstream and intergenic gene variations in K133PMM. There were 259 single nucleotide polymorphisms (SNPs), 187 insertion-deletion (InDels), and 546 copy number variations (CNVs) identified. Most of the genomic variations were found in the gene's upstream and non-coding regions. Ploidy estimation revealed chromosome 5 in tetrasomy and 6, 9, and 12 in trisomy, uniquely in K133PMM. These contain the genes for protein degradation, parasite motility, autophagy, cell cycle maintenance, and drug efflux membrane transporters. Furthermore, we also observed reduction in ploidy of chromosomes 15, 20, and 23, in the resistant parasite containing mostly the genes for hypothetical proteins and membrane transporters. We chronicled correlated genomic conversion and aneuploidy in parasites and hypothesize that this led to rapid evolutionary changes in response to drug induced pressure, which causes them to become resistant. [ABSTRACT FROM AUTHOR]

Leishmaniasis on the Indian subcontinent is thought to have an anthroponotic transmission cycle. There is no direct evidence that a mammalian host other than humans can be infected with Leishmania donovani and transmit infection to the sand fly vector. The aim of the present study was to evaluate the impact of sand fly feeding on other domestic species and provide clinical evidence regarding possible non-human reservoirs through experimental sand fly feeding on cows, water buffalo goats and rodents. We performed xenodiagnosis using colonized Phlebotomus argentipes sand flies to feed on animals residing in villages with active Leishmania transmission based on current human cases. Xenodiagnoses on mammals within the endemic area were performed and blood-fed flies were analyzed for the presence of Leishmania via qPCR 48hrs after feeding. Blood samples were also collected from these mammals for qPCR and serology. Although we found evidence of Leishmania infection within some domestic mammals, they were not infectious to vector sand flies. Monitoring infection in sand flies and non-human blood meal sources in endemic villages leads to scientific proof of exposure and parasitemia in resident mammals. Lack of infectiousness of these domestic mammals to vector sand flies indicates that they likely play no role, or a very limited role in Leishmania donovani transmission to people in Bihar. Therefore, a surveillance system in the peri-/post-elimination phase of visceral leishmaniasis (VL) must monitor absence of transmission. Continued surveillance of domestic mammals in outbreak villages is necessary to ensure that a non-human reservoir is not established, including domestic mammals not present in this study, specifically dogs. Author summary: Leishmania donovani, the causative agent of visceral leishmaniasis (VL), has unique enzootic, zoonotic and anthroponotic life cycles dependent on geographic region. Most Leishmania spp. are zoonotic, transmitted between humans and non-human mammals. Leishmaniasis is endemic in over 98 countries and the estimated population at risk on the Indian sub-continent (ISC) is around 0.2–0.4 million people. Herein, we assess knowledge gaps in disease transmission that challenge dogma regarding the reservoir(s) of visceral leishmaniasis and control efforts on the Indian sub-continent. Better understanding of L. donovani infection in domestic animals and its transmission to sand flies will provide answers to fundamental questions in VL epidemiology and ecology. Blood samples were collected from livestock and rodents in endemic villages and colonized sand flies were fed on these animals and analyzed post feeding to see if they could pick up parasites via bloodmeal. We found that livestock and to a smaller extent rodents could be infected with or exposed to Leishmania infection. However, these mammals were not infectious to Phlebotomus argentipes, the predominant vector species incriminated as transmitting L. donovani in India. The inability of these domestic mammals to transmit L. donovani to sand flies in Bihar suggests that they play a limited role in the spread of infection. A key component in reducing and preventing the re-emergence of VL in the context of the elimination program is to understand the role of vector feeding on non-human animals in the transmission cycle. Better estimation of the proportion of the livestock and rodent population exposed to sand flies during different seasons and ensuring that there are not reservoir species that contribute to transmission will help implement appropriate control strategies for sustainable elimination. [ABSTRACT FROM AUTHOR]

Vectors are biological organisms that can spread infectious pathogens from animals to individuals or between individuals. Human health issues are called vector-borne diseases when germs, parasites, and viruses are released by vectors. The presence of these diseases is extreme in the tropical and subtropical zone, and they mainly affect the poorest populations. Every year there are more than 700,000 deaths occurred by Vector-borne diseases. Leishmaniasis is an extensively transmitted protozoan vector-borne disease and it has been one of the most neglected tropical diseases for decades. Leishmania donovani and Leishmania infantum have been the most common species out of 20 intracellular protozoan parasites of genus Leishmania that causes visceral leishmaniasis (VL), the deadliest form. Due to the lack of a potent vaccine and effective vector control plan, chemotherapy has been considered a prime tool for the treatment of leishmaniasis. The antileishmanial drugs widely used are highly toxic, injectable (painful and require hospitalization), very expensive, and inferior efficacy for HIV co-infected VL patients. At present antimonials come in first-line therapy and failure of which the second-line drug Amphotericin B is generally considered. Miltefosine, an antineoplastic agent, had been emerged as the only oral anti-leishmanial drug. Unfortunately, the parasites introduce unavoidably resistance toward most of these antileishmanial drugs. Many resistance mechanisms that arise by these parasites are associated with a minimum uptake or increased efflux of the drug due to modification or altered expression of membrane transporters. Therefore, up-to-date strategies are required to discover more effective and less toxic nanomedicines that could specifically solve drug resistance associated with Leishmania chemotherapy. The Leishmania parasites completely contaminate highly phagocytic cells (macrophages), this aspect can be utilized for the targeted delivery formulation of nanoparticles. A number of nanotechnology-based approaches and products have come to light as anti-leishmanial drugs. These approaches are polymeric nanoparticles, liposomes, metallic oxide nanoparticles, lipid nano-capsules, nanosuspension, nanotube, and nano vaccines. These nanoparticles have exclusive inherent properties such as large pore size, high surface area, good biocompatibility and biodegradability, stable aqueous dispersion, etc. This review offers an overview of the present leishmania disease situation globally, its traditional pharmacological treatments, and how the usage of various nanoparticles can help to solve the issue of drug resistance. [ABSTRACT FROM AUTHOR]