Effect of Leishmania infantum infection on B cell lymphopoiesis and memory in the bone marrow and spleen.

Visceral leishmaniasis (VL) is characterized by an uncontrolled infection of internal organs such as the spleen, liver and bone marrow (BM) and can be lethal when left untreated. No effective vaccination is currently available for humans. The importance of B cells in infection and VL protective immunity has been controversial, with both detrimental and protective effects described. VL infection was found in this study to increase not only all analyzed B cell subsets in the spleen but also the B cell progenitors in the BM. The enhanced B lymphopoiesis aligns with the clinical manifestation of polyclonal hypergammaglobulinemia and the occurrence of autoantibodies. In line with earlier reports, flow cytometric and microscopic examination identified parasite attachment to B cells of the BM and spleen without internalization, and transformation of promastigotes into amastigote morphotypes. The interaction appears independent of IgM expression and is associated with an increased detection of activated lysosomes. Furthermore, the extracellularly attached amastigotes could be efficiently transferred to infect macrophages. The observed interaction underscores the potentially crucial role of B cells during VL infection. Additionally, using immunization against a fluorescent heterologous antigen, it was shown that the infection does not impair immune memory, which is reassuring for vaccination campaigns in VL endemic areas.

Visceral Leishmaniasis-Human Immunodeficiency Virus-Coinfected Patients Are Highly Infectious to Sandflies in an Endemic Area in India.

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.

Disease-specific differences in pharmacokinetics of paromomycin and miltefosine between post-kala-azar dermal leishmaniasis and visceral leishmaniasis patients in eastern Africa.

Treatment regimens for post-kala-azar dermal leishmaniasis (PKDL) are usually extrapolated from those for visceral leishmaniasis (VL), but drug pharmacokinetics (PK) can differ due to disease-specific variations in absorption, distribution, and elimination. This study characterized PK differences in paromomycin and miltefosine between 109 PKDL and 264 VL patients from eastern Africa. VL patients showed 0.55-fold (95%CI: 0.41-0.74) lower capacity for paromomycin saturable reabsorption in renal tubules, and required a 1.44-fold (1.23-1.71) adjustment when relating renal clearance to creatinine-based eGFR. Miltefosine bioavailability in VL patients was lowered by 69% (62-76) at treatment start. Comparing PKDL to VL patients on the same regimen, paromomycin plasma exposures were 0.74-0.87-fold, while miltefosine exposure until the end of treatment day was 1.4-fold. These pronounced PK differences between PKDL and VL patients in eastern Africa highlight the challenges of directly extrapolating dosing regimens from one leishmaniasis presentation to another.

IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans.

The intracellular protozoan parasite Leishmania donovani causes debilitating human diseases that involve visceral and dermal manifestations. Type 3 interferons (IFNs), also referred to as lambda IFNs (IFNL, IFN-L, or IFN-λ), are known to play protective roles against intracellular pathogens at the epithelial surfaces. Herein, we show that L. donovani induces IFN-λ3 in human as well as mouse cell line-derived macrophages. Interestingly, IFN-λ3 treatment significantly decreased parasite load in infected cells, mainly by increasing reactive oxygen species production. Microscopic examination showed that IFN-λ3 inhibited uptake but not replication, while the phagocytic ability of the cells was not affected. This was confirmed by experiments that showed that IFN-λ3 could decrease parasite load only when added to the medium at earlier time points, either during or soon after parasite uptake, but had no effect on parasite load when added at 24 h post-infection, suggesting that an early event during parasite uptake was targeted. Furthermore, the parasites could overcome the inhibitory effect of IFN-λ3, which was added at earlier time points, within 2-3 days post-infection. BALB/c mice treated with IFN-λ3 before infection led to a significant increase in expression of IL-4 and ARG1 post-infection in the spleen and liver, respectively, and to different pathological changes, especially in the liver, but not to changes in parasite load. Treatment with IFN-λ3 during infection did not decrease the parasite load in the spleen either. However, IFN-λ3 was significantly increased in the sera of visceral leishmaniasis patients, and the IFNL genetic variant rs12979860 was significantly associated with susceptibility to leishmaniasis.

Chronic High-Level Parasitemia in HIV-Infected Individuals With or Without Visceral Leishmaniasis in an Endemic Area in Northwest Ethiopia: Potential Superspreaders?

BACKGROUND: People with human immunodeficiency virus (PWH) with recurrent visceral leishmaniasis (VL) could potentially drive Leishmania transmission in areas with anthroponotic transmission such as East Africa, but studies are lacking. Leishmania parasitemia has been used as proxy for infectiousness. METHODS: This study is nested within the Predicting Visceral Leishmaniasis in HIV-InfectedPatients (PreLeisH) prospective cohort study, following 490 PWH free of VL at enrollment for up to 24-37 months in northwest Ethiopia. Blood Leishmania polymerase chain reaction (PCR) was done systematically. This case series reports on 10 PWH with chronic VL (≥3 VL episodes during follow-up) for up to 37 months, and 3 individuals with asymptomatic Leishmania infection for up to 24 months. RESULTS: All 10 chronic VL cases were male, on antiretroviral treatment, with 0-11 relapses before enrollment. Median baseline CD4 count was 82 cells/µL. They displayed 3-6 VL treatment episodes over a period up to 37 months. Leishmania blood PCR levels were strongly positive for almost the entire follow-up (median cycle threshold value, 26 [interquartile range, 23-30]), including during periods between VL treatment. Additionally, we describe 3 PWH with asymptomatic Leishmania infection and without VL history, with equally strong Leishmania parasitemia over a period of up to 24 months without developing VL. All were on antiretroviral treatment at enrollment, with baseline CD4 counts ranging from 78 to 350 cells/µL. CONCLUSIONS: These are the first data on chronic parasitemia in PWH from Leishmania donovani-endemic areas. PWH with asymptomatic and symptomatic Leishmania infection could potentially be highly infectious and constitute Leishmania superspreaders. Xenodiagnosis studies are required to confirm infectiousness.

The Hidden Hand of Asymptomatic Infection Hinders Control of Neglected Tropical Diseases: A Modeling Analysis.

BACKGROUND: Neglected tropical diseases are responsible for considerable morbidity and mortality in low-income populations. International efforts have reduced their global burden, but transmission is persistent and case-finding-based interventions rarely target asymptomatic individuals. METHODS: We develop a generic mathematical modeling framework for analyzing the dynamics of visceral leishmaniasis in the Indian sub-continent (VL), gambiense sleeping sickness (gHAT), and Chagas disease and use it to assess the possible contribution of asymptomatics who later develop disease (pre-symptomatics) and those who do not (non-symptomatics) to the maintenance of infection. Plausible interventions, including active screening, vector control, and reduced time to detection, are simulated for the three diseases. RESULTS: We found that the high asymptomatic contribution to transmission for Chagas and gHAT and the apparently high basic reproductive number of VL may undermine long-term control. However, the ability to treat some asymptomatics for Chagas and gHAT should make them more controllable, albeit over relatively long time periods due to the slow dynamics of these diseases. For VL, the toxicity of available therapeutics means the asymptomatic population cannot currently be treated, but combining treatment of symptomatics and vector control could yield a quick reduction in transmission. CONCLUSIONS: Despite the uncertainty in natural history, it appears there is already a relatively good toolbox of interventions to eliminate gHAT, and it is likely that Chagas will need improvements to diagnostics and their use to better target pre-symptomatics. The situation for VL is less clear, and model predictions could be improved by additional empirical data. However, interventions may have to improve to successfully eliminate this disease.

New synthetic molecules incorporated into polymeric micelles used for treatment against visceral leishmaniasis.

Treatment against visceral leishmaniasis (VL) presents problems, mainly related to drug toxicity, high cost and/or by emergence of resistant strains. In the present study, two vanillin synthetic derivatives, 3 s [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] and 3 t [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde], were evaluated as therapeutic candidates in a murine model against Leishmania infantum infection. Molecules were used pure (3 s and 3 t) or incorporated into Poloxamer 407-based micelles (3 s/M and 3 t/M) in the infected animals, which also received amphotericin B (AmpB) or Ambisome® as control. Results showed that 3 s/M and 3 t/M compositions induced a Th1-type immune response in treated animals, with higher levels of IFN-γ, IL-2, TNF-α, IL-12, nitrite, and IgG2a antibodies. Animals presented also low toxicity and significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, as compared as control groups mice, with the evaluations performed one and 30 days after the application of the therapeutics. In conclusion, preliminary data suggest that 3 s/M and 3 t/M could be considered for future studies as therapeutic agents against VL.

A case-control study on risk factors for visceral leishmaniasis in West Pokot County, Kenya.

BACKGROUND: Visceral leishmaniasis (VL) is a severe parasitic disease transmitted by phlebotomine sandflies. VL is endemic in West Pokot County, Kenya, where effective strategies to interrupt transmission are impeded by the limited understanding of VL risk factors. Therefore, this case-control study aimed to explore environmental, behavioural and household determinants of VL in West Pokot. METHODS: From November 2022 to January 2023, a structured questionnaire was administered to 36 symptomatic primary VL cases attending Kacheliba Sub-County Hospital in West Pokot and to 50 healthy controls from local villages. The VL status of all participants was confirmed using an rK39 rapid diagnostic test. Associations between questioned determinants and VL were investigated by means of age-corrected univariate logistic regression analysis. RESULTS: Significant associations were found between VL and housing characteristics, such as window presence and floor type. VL cases more frequently reported the presence of cattle, dogs and sheep in their house yards. VL was also associated with cutting down trees in the house yard and house proximity to several Acacia tree species. Furthermore, outdoor activities, including travelling outside the residence for more than 2 weeks, activities near termite mounds, and forest activities during the rainy season, increased the risk of VL. CONCLUSIONS: This work reports a number of previously undescribed risk factors for VL in the understudied West Pokot focus. The results suggest VL transmission occurs both peri-domestically at night and outdoors during the day, particularly when sandfly resting sites are disturbed. Our findings warrant further research into sandfly ecology and potential zoonotic parasite reservoirs in West Pokot.

Serological characteristics and clinical implications of IgG subclasses in visceral leishmaniasis.

OBJECTIVES: Visceral leishmaniasis (VL) represents the most severe form of Leishmaniasis infection, often resulting in fatality without timely treatment. Previous studies have found that immunosuppression increases the risk of VL disease progression and mortality, and the total immunoglobulin G (IgG) levels in peripheral blood vary before and after treatment. However, the distinct levels and roles of IgG subclasses in VL have not been documented yet. This study aims to elucidate the characteristics and clinical significance of IgG subclasses in VL. METHODS: A total of 43 cases newly-diagnosed with VL were enrolled in the cohort. We measured the levels of IgG subclasses before and after standard treatment and conducted assessments of bone marrow features. In addition, we analysed other haematological indices and examined the variations in IgG subclasses, as well as their correlation with clinical and laboratory factors. RESULTS: The levels of total IgG, IgG1, and the ratios of both IgG1/IgG and IgG1/IgG2 decreased significantly after treatment, whereas the ratios of IgG2/ IgG showed an obvious increase. The VL patients without hyperglobulinemia displayed significant lower IgG1/IgG2 ratios, but higher IgG2/IgG ratios compared with those with hyperglobulinemia. In addition, VL patients with positive bone marrow amastigotes had significant higher IgG1/IgG and IgG1/IgG2 ratios, but lower IgG2/IgG ratios. IgG subclasses were correlated with abnormal blood test results, particularly immunological elements including IgM and Complement 4 (C4). CONCLUSIONS: IgG1 and IgG2 exhibited contrasting changes after treatment in VL patients. The features of bone marrow and laboratory tests indicated that IgG1 and IgG2 serve different roles in the progression of VL. The ratios of IgG subclasses may be more precise indicators to evaluate immune reaction in VL than traditional total IgG.

Secondary hemophagocytic lymphohistiocytosis in pediatric patients with visceral leishmaniasis and Epstein-Barr virus infection.

Visceral leishmaniasis-associated hemophagocytic lymphohistiocytosis (VL-HLH) is indistinguishable from those of HLH of other etiologies due to the overlap symptoms, posing a serious threat to life. In this study, we aimed to provide insights for early diagnosis and improve outcomes in pediatric patients with VL-HLH. We retrospectively analyzed the clinical and laboratory data of 10 pediatric patients with VL-HLH and 58 pediatric patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH). The median time from symptom onset to cytopenia in patients with VL-HLH and EBV-HLH was 11 days (interquartile range, 7-15 days) and five days (interquartile range, 3.75-9.25 days) (P = 0.005). Both groups showed liver injury and increased lactate dehydrogenase levels; however the levels of aspartate aminotransferase, alanine aminotransferase, direct bilirubin, and lactate dehydrogenase in patients with VL-HLH were significantly lower than those in patients with EBV-HLH (P < 0.05). The fibrinogen and triglyceride levels were almost normal in VL-HLH patients but were significantly altered in EBV-HLH cases ( P < 0.05). The positive rate of first bone marrow microscopy examination, anti-rK39 IgG detection, and blood metagenomic next-generation sequencing was 50%, 100%, and 100%, respectively. After VL diagnosis, eight patients were treated with sodium stibogluconate and two were treated with liposomal amphotericin B. All the patients with VL-HLH recovered. Our study demonstrates that regular triglyceride and fibrinogen levels in pediatric patients with VL-HLH may help in differential diagnosis from EBV-HLH. VL-HLH is milder than EBV-HLH, with less severe liver injury and inflammatory responses, and timely treatment with antileishmanial agents is essential to improve the outcomes of pediatric patients with VL-HLH.

LaSap vaccine: Immunotherapy and immunochemotherapy associated with allopurinol in dogs naturally infected with Leishmania infantum.

Canine visceral leishmaniasis is a parasitic zoonosis that has a profound impact on public health in countries where it is endemic. Chemotherapeutic treatments cannot keep dogs stable for long periods, and the risk of generating parasitic resistance must be considered. Forty-four symptomatic and naturally infected dogs with Leishmania infantum were tested with two treatment protocols (i) immunotherapy with LaSap vaccine and (ii) immunochemotherapy with LaSap vaccine plus allopurinol. At 90 days after the end of the treatment, it was verified that, although both protocols had generated significant clinical improvements with a greater production of IFN-γ/IL-10, in relation to the parasite load, mainly in the skin, the dogs treated only with immunotherapy maintained the same profile. These results indicate that LaSap is a good strategy to control dog parasitism.

Apolipoprotein E Is Upregulated in Blood and Circulating Monocytes of Indian Patients With Visceral Leishmaniasis.

Apolipoprotein E (ApoE) has been associated with several diseases including Parkinson's disease, Alzheimer's and multiple sclerosis. ApoE also has documented immunomodulatory functions. We investigated gene expression in circulating monocytes and in bone marrows of patients with visceral leishmaniasis (VL) living in an endemic area in Bihar, India, and contrasted these with control healthy subjects or other diagnostic bone marrows from individuals in the same region. Samples from VL patients were obtained prior to initiating treatment. Our study revealed significant upregulated expression of the apoE transcript in patients with VL. Furthermore, the levels of ApoE protein were elevated in serum samples of subjects with VL compared with healthy endemic controls. These observations may provide clues regarding the complex interactions between lipid metabolism and immunoregulation of infectious and inflammatory diseases.

Understanding hypergammaglobulinemia in experimental or natural visceral leishmaniasis.

Nonspecific hypergammaglobulinemia (HGG) occurs in symptomatic human visceral leishmaniasis (VL) caused by L. L. infantum. This study assessed this finding in experimental infection in hamsters and natural infection in dogs. The serum concentration of proteins, albumin and globulins was determined through the biuret and bromocresol green reaction, where the HGG was better expressed through the albumin/globulin (A/G) ratio. HGG was associated with a higher concentration of specific anti-glycan antibodies (BSA-G)/promastigote soluble extract (PSE) and the presence of circulating immune complexes (IC) by dissociative enzyme-linked immunoassay (ELISA). The study found monovalent IC in 37.9% (PSE) and 50% (BSA-G) of sera from infected hamsters, with increased frequency as the disease progressed. HGG was found in >60% of the samples in dogs with VL, associated with higher levels of specific immunoglobulin (Ig)A and IgM, but not IgG, determined using the PSE and BSA-G ELISA. HGG was associated with the presence of monovalent IC in 58.9% (PSE) and 63.4% (BSA-G) positive dog samples. HGG may result not only from the nonspecific activation of B cells, with greater production of specific and nonspecific antibodies, but also due to lower IgG excretion due to the presence of soluble monovalent IC. HGG correlates to the progression of VL and may be a marker for manifested disease.

Immunotherapy Combining Mimotopes Selected by Phage Display Plus Amphotericin B Is Effective for Treatment Against Visceral Leishmaniasis.

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.

Portable smartphone-based molecular test for rapid detection of Leishmania spp.

PURPOSE: Leishmaniasis, caused by the parasite of the genus Leishmania, is a neglected tropical disease which is endemic in more than 60 countries. In South-East Asia, Brazil, and East Africa, it mainly occurs as kala-azar (visceral leishmaniasis, VL), and subsequently as post kala-azar dermal leishmaniasis (PKDL) in a smaller portion of cases. As stated per WHO roadmap, accessibility to accurate diagnostic methods is an essential step to achieve elimination. This study aimed to test the accuracy of a portable minoo device, a small battery-driven, multi-use fluorimeter operating with isothermal technology for molecular diagnosis of VL and PKDL. METHODS: Fluorescence data measured by the device within 20 min are reported back to the mobile application (or app) via Bluetooth and onward via the internet to a backend. This allows anonymous analysis and storage of the test data. The test result is immediately returned to the app displaying it to the user. RESULTS: The limit of detection was 11.2 genome copies (95% CI) as determined by screening a tenfold dilution range of whole Leishmania donovani genomes using isothermal recombinase polymerase amplification (RPA). Pathogens considered for differential diagnosis were tested and no cross-reactivity was observed. For its diagnostic performance, DNA extracted from 170 VL and PKDL cases, comprising peripheral blood samples (VL, n = 96) and skin biopsies (PKDL, n = 74) from India (n = 108) and Bangladesh (n = 62), was screened. Clinical sensitivity and specificity were 88% and 91%, respectively. CONCLUSION: Minoo devices can offer a convenient, cheaper alternative to other molecular diagnostics. Its easy handling makes it ideal for use in low-resource settings to identify parasite burden.

Cytokine profiles, blood parasite load and clinical features of visceral leishmaniasis in West Pokot County, Kenya.

Visceral leishmaniasis (VL) is a severe infectious disease caused by protozoan parasites of the Leishmania donovani complex. Blood cytokine concentrations in VL patients can inform us about underlying immunopathogenesis and may serve as a biomarker for treatment effectiveness. However, cytokine levels have not yet been studied in VL patients from Kenya, where case load is high. This study measured the serum cytokine profile, blood parasite load and clinical and haematological features of VL patients from West Pokot County, Kenya, over the course of treatment with sodium stibogluconate and paromomycin (SSG-PM). VL patients recruited at the hospital presented with splenomegaly and weight loss, and frequently had pancytopenia and anaemia. Median Leishmania parasite load in blood, determined with real-time polymerase chain reaction, was 2.6 × 104 parasite equivalents mL−1. Compared to endemic healthy controls, serum interferon gamma (IFN-γ), interleukin 5 (IL-5), IL-6, IL-10, IL-12p70, IL-17A and IL-27 were significantly elevated in untreated VL patients. Severe VL was associated with higher IL-10 and lower IFN-γ levels. After 17 daily injections with SSG-PM, disease symptoms disappeared, leukocyte and thrombocyte counts significantly increased, and blood parasite load decreased to undetectable levels in all VL patients. There was a significant decrease in IL-10 and IL-6, whereas IL-17A levels increased; the remaining cytokines showed no significant concentration change during treatment. In conclusion, the results suggest that SSG-PM treatment of VL patients from West Pokot was effective. Moreover, both inflammatory and regulatory immune responses appeared to decrease during treatment, although the increase in IL-17A could reflect a partial continuation of immune activation.

Insights into nucleoside hydrolase from Leishmania donovani inhibition: A new bioaffinity chromatography-based screening assay and docking studies.

Leishmaniasis, a group of neglected infectious diseases, encompasses a serious health concern, particularly with visceral leishmaniasis exhibiting potentially fatal outcomes. Nucleoside hydrolase (NH) has a fundamental role in the purine salvage pathway, crucial for Leishmania donovani survival, and presents a promising target for developing new drugs for visceral leishmaniasis treatment. In this study, LdNH was immobilized into fused silica capillaries, resulting in immobilized enzyme reactors (IMERs). The LdNH-IMER activity was monitored on-flow in a multidimensional liquid chromatography system, with the IMER in the first dimension. A C18 analytical column in the second dimension furnished the rapid separation of the substrate (inosine) and product (hypoxanthine), enabling direct enzyme activity monitoring through product quantification. LdNH-IMER exhibited high stability and was characterized by determining the Michaelis-Menten constant. A known inhibitor (1-(ß-d-Ribofuranosyl)-4-quinolone derivative) was used as a model to validate the established method in inhibitor recognition. Screening of three additional derivatives of 1-(ß-d-Ribofuranosyl)-4-quinolone led to the discovery of novel inhibitors, with compound 2a exhibiting superior inhibitory activity (Ki = 23.37 ± 3.64 µmol/L) compared to the employed model inhibitor. Docking and Molecular Dynamics studies provided crucial insights into inhibitor interactions at the enzyme active site, offering valuable information for developing new LdNH inhibitors. Therefore, this study presents a novel screening assay and contributes to the development of potent LdNH inhibitors.

Exploring host epigenetic enzymes as targeted therapies for visceral leishmaniasis: in silico design and in vitro efficacy of KDM6B and ASH1L inhibitors.

In order to combat various infectious diseases, the utilization of host-directed therapies as an alternative to chemotherapy has gained a lot of attention in the recent past, since it bypasses the existing limitations of conventional therapies. The use of host epigenetic enzymes like histone lysine methyltransferases and lysine demethylases as potential drug targets has successfully been employed for controlling various inflammatory diseases like rheumatoid arthritis and acute leukemia. In our earlier study, we have already shown that the functional knockdown of KDM6B and ASH1L in the experimental model of visceral leishmaniasis has resulted in a significant reduction of organ parasite burden. Herein, we performed a high throughput virtual screening against KDM6B and ASH1L using > 53,000 compounds that were obtained from the Maybridge library and PubChem Database, followed by molecular docking to evaluate their docking score/Glide Gscore. Based on their docking scores, the selected inhibitors were later assessed for their in vitro anti-leishmanial efficacy. Out of all inhibitors designed against KDM6B and ASH1L, HTS09796, GSK-J4 and AS-99 particularly showed promising in vitro activity with IC50 < 5 µM against both extracellular promastigote and intracellular amastigote forms of L. donovani. In vitro drug interaction studies of these inhibitors further demonstrated their synergistic interaction with amphotericin-B and miltefosine. However, GSK-J4 makes an exception by displaying an in different mode of interaction with miltefosine. Collectively, our in silico and in vitro studies acted as a platform to identify the applicability of these inhibitors targeted against KDM6B and ASH1L for anti-leishmanial therapy.

In vitro growth of Leishmania parasites from biopsy samples of suspected cutaneous and visceral leishmaniasis cases in Sri Lanka: An observational study.

Sri Lanka reports a large focus of Leishmania donovani caused cutaneous leishmaniasis (CL). Subsequent emergence of visceral leishmaniasis (VL) was also reported recently. Expansion of the on-going disease outbreak and many complexities indicate urgent need to enhance early case detection methods. In vitro cultivation (IVC) of parasites causing visceral leishmaniasis (VL) is important for disease confirmation and to obtain sufficient quantities of parasites required in many scientific studies. IVC is carried out as a useful second line investigation for direct microscopy negative patients with CL in this setting. Along with the emergence of VL, current study was carried out to evaluate in vitro growth of local VL parasites and to identify their differences associated with in vitro growth characteristics. Routine parasitological diagnostic methods, i.e., light microscopy (LM), polymerase chain reaction (PCR) were used for confirmation of suspected cases. Lesion samples from 125 suspected CL cases and bone marrow or splenic aspirations from 125 suspected VL patients were used to inoculate IVCs. Media M199 (about 70 µl) supplemented with 15-20% of heat inactivated fetal bovine serum was used for initial culturing procedures in capillaries. Capillary cultures were monitored daily. Total of 44 different compositions/conditions were used for evaluating in vitro growth of VL causing parasite. Daily records on parasite counts, morphological appearance (size, shape, and wriggly movements) were maintained. In vitro transformation of Leishmania promastigotes to amastigotes and outcome of the attempts on recovery of live Leishmania from culture stabilates was also compared between CL and VL parasites. Proportion of cultures showing a transformation of promastigotes were 40/45 (88.9%) and 4/10 (40.0%) for CL and VL respectively. In the transformed cultures, parasites showing typical shape, size and movement patterns were less in VL (1/4, 25.0%) compared to CL (28/40, 70.0%). CL cultures showed a growth up to mass culturing level with mean duration of two weeks while it was about five weeks for VL cultures. Proportion of cultures that reached a parasite density of 1 × 106 cells/ml (proceeded to mass cultures) was significantly low in VL (4/10, 40%) as compared to CL (28/40, 70.0%). None of media compositions/conditions were successful for mass culturing of VL parasites while all of them were shown to be useful for growing CL strains. Also in vitro transformation to amastigote form and recovering of culture stabilates were not successful compared to CL. There were clear differences between in vitro growth of Leishmania parasites causing local CL and VL. Further studies are recommended for optimization of in vitro culturing of VL parasite which will be invaluable to enhance case detection in future.

A review of non-invasive samples and tools in kala-azar diagnosis and test of cure.

The recurrence of visceral leishmaniasis (VL), also called kala-azar (KA), in endemic regions of tropical countries like India, is primarily attributed to asymptomatic VL, post-kala azar dermal leishmaniasis (PKDL), and human immunodeficiency virus (HIV) co-infection. To effectively manage VL cases and elimination targets, an early and rapid diagnosis as well as accurate field surveillance is highly essential. The traditional sampling methods like bone marrow (BM), spleen, and lymph node (LN) tissue aspirations are invasive, painful, tedious, and prone to nosocomial infections, require skilled persons and hospital facilities, and are not feasible in rural areas. Therefore, there is an urgent requirement for the adoption of a patient-friendly, non-invasive, non-hospitalized sampling procedure that ensures an effective VL diagnosis. This review aims to meticulously evaluate the most recent scientific research that focuses on the precision, feasibility, and applicability of non-invasive sampling (NIS) and techniques for the diagnosis and test of cure of VL, particularly in resource-limited settings. Apart from that, the non-invasive techniques (NIT) that have shown promising results while monitoring VL treatment response and relapse are also reviewed. The limitations associated with NIT and possible improvements in this regard are discussed as well to improve the diagnosis and management of VL.