Active Visceral Leishmaniasis Research Articles

Altered IL-7 signaling in CD4+ T cells from patients with visceral leishmaniasis.

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. 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. 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.

A case report on para-kala-azar dermal leishmaniasis: an unresolved mystery

BackgroundPost-kala-azar dermal leishmaniasis (PKDL) is a dermatosis that occurs 2–3 years after an apparently successful treatment of visceral leishmaniasis (VL). In rare cases, PKDL occurs concurrently with VL and is characterized by fever, splenomegaly, hepatomegaly or lymphadenopathy, and poor nutritional status and is known as Para-kala-azar dermal leishmaniasis (Para-KDL). Co-association of active VL in PKDL patients is documented in Africa, but very few case reports are found in South Asia.Case summaryWe present a case of Para-kala-azar Dermal Leishmaniasis (Para-KDL) in a 50-year-old male patient with a history of one primary Visceral Leishmaniasis (VL) and 2 times relapse of Visceral Leishmaniasis (VL). The patient presented with fever, skin lesions, and hepatosplenomegaly. Laboratory tests revealed LD bodies in the slit skin smear and splenic biopsy. The patient was treated with two cycles of Amphotericin B with Miltefosine in between cycles for 12 weeks to obtain full recovery.ConclusionThis case report serves as a reminder that Para-kala-azar dermal leishmaniasis can develop as a consequence of prior visceral leishmaniasis episodes, even after apparently effective therapy. Since para-kala-azar is a source of infectious spread, endemics cannot be avoided unless it is effectively recognized and treated.

Prediction of an immunogenic peptide ensemble and multi-subunit vaccine for Visceral leishmaniasis using bioinformatics approaches

Visceral Leishmaniasis (VL) is a neglected tropical disease of public health importance in the Indian subcontinent. Despite consistent elimination initiatives, the disease has not yet been eliminated and there is an increased risk of resurgence from active VL reservoirs including asymptomatic, post kala azar dermatitis leishmaniasis (PKDL) and HIV-VL co-infected individuals. To achieve complete elimination and sustain it in the long term, a prophylactic vaccine, which can elicit long lasting immunity, is desirable. In this study, we employed immunoinformatic tools to design a multi-subunit epitope vaccine for the Indian population by targeting antigenic secretory proteins screened from the Leishmania donovani proteome. Out of 8014 proteins, 277 secretory proteins were screened for their cellular location and proteomic evidence. Through NCBI BlastP, unique fragments of the proteins were cropped, and their antigenicity was evaluated. B-cell, HTL and CTL epitopes as well as IFN-ɣ, IL-17, and IL-10 inducers were predicted, manually mapped to the fragments and common regions were tabulated forming a peptide ensemble. The ensemble was evaluated for Class I MHC immunogenicity and toxicity. Further, immunogenic peptides were randomly selected and used to design vaccine constructs. Eight vaccine constructs were generated by linking random peptides with GS linkers. Synthetic TLR-4 agonist, RS09 was used as an adjuvant and linked with the constructs using EAAK linkers. The predicted population coverage of the constructs was ∼99.8 % in the Indian as well as South Asian populations. The most antigenic, nontoxic, non-allergic construct was chosen for the prediction of secondary and tertiary structures. The 3D structures were refined and analyzed using Ramachandran plot and Z-scores. The construct was docked with TLR-4 receptor. Molecular dynamic simulation was performed to check for the stability of the docked complex. Comparative in silico immune simulation studies showed that the predicted construct elicited humoral and cell-mediated immunity in human host comparable to that elicited by Leish-F3, which is a promising vaccine candidate for human VL.

Total serum N-glycans mark visceral leishmaniasis in human infections with Leishmania infantum

Visceral leishmaniasis (VL) is a clinical form of leishmaniasis with high mortality rates when not treated. Diagnosis suffers from invasive techniques and sub-optimal sensitivities. The current (affordable) treatment with pentavalent antimony as advised by the WHO is possibly harmful to the patient. There is need for an improved diagnosis to prevent possibly unnecessary treatment. N-glycan analysis may aid in diagnosis. We evaluated the N-glycan profiles from active VL, asymptomatic infections (ASYMP) and controls from non-endemic (NC) and endemic (EC) areas. Active VL has a distinct N-glycome profile that associates with disease severity. Our study suggests that the observed glycan signatures could be a valuable additive to diagnosis and assist in identifying possible markers of disease and understanding the pathogenesis of VL. Further studies are warranted to assess a possible future role of blood glycome analysis in active VL diagnosis and should aim at disease specificity.

CD4+ T cells show a cytolytic phenotype functions in human visceral leishmaniasis

Abstract Control of Visceral leishmaniasis (VL), a parasitic disease caused by L. donovani, requires robust CD4 +T cells response to control the parasite replication by IFN-γ production and activation of macrophages. Our recent study on transcriptional signature of CD4 +T cell isolated from peripheral blood of active VL patients showed enhanced expression of genes related to cytotoxicity. In present study, we investigated expression of these cytotoxic molecules including granzyme B (GZMB), Granulysin, Perforin and NKG7 on different CD4 +T cell subsets to establish the role of GZMB in regulating CD4 +T cells functional capacity. PBMCs cells were isolated from active and post treated VL patients and endemic controls, and flow cytometry was performed. LAMP assay (CD107b expression) was performed to study the degranulation capacity of CD4 +T cells. Whole blood assay followed by ELISA was performed to measure antigen specific cytokine / GZMB production by CD4 +T cells. We found that activated and degranulating CD4 +T cells (CD38 +CD107 +) had higher expression of GZMB, Granulysin, perforin and NKG-7. Similarly, GZMB level in plasma and antigen stimulated whole blood culture supernatant were significantly elevated in active VL patient compared to post treatment and EC. There was no change is IFN-γ secretion when whole blood culture was stimulated with soluble leishmania antigen in presence of Con-A but a significant decline in IL-6 production was observed. In summary, VL CD4 +T cells show a cytolytic phenotype and further investigations are required to understand their differentiation and function, particularly for promoting anti-parasitic immunity for host protection and effective intervention or therapy. Extramural Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (TMRC grant number U19AI074321)

TREM-1 Expression on the Surface of Neutrophils in Patients With Visceral Leishmaniasis Is Associated With Immunopathogenesis.

Visceral leishmaniasis (VL) is a systemic chronic and potentially fatal disease for humans. Mechanisms related to the dysregulation of the inflammatory response may be involved in both the pathogenesis and prognosis of VL. Triggering Receptor Expressed on Myeloid Cells-1 (TREM-1) is a receptor constitutively expressed on neutrophils and monocyte subsets. The protein serves to regulate and amplify inflammatory responses. This study aimed to evaluate the expression profile of TREM-1 on the surface of neutrophils from patients with VL at varying time points during leishmanicidal treatment. For this purpose, neutrophils were isolated from the peripheral blood of patients with VL at different stages of treatment, which include 0, 7, and 30 days after treatment. Surface TREM-1 expression was assessed by immunophenotyping neutrophil populations. In addition, the association of TREM-1 expression on the surface of neutrophils with clinical and laboratory parameters and serum levels of inflammatory mediators was also evaluated. Results demonstrate a lower surface expression of TREM-1 in VL patients in the absence of treatment. However, increased levels of TREM-1 expression were observed 7 and 30 days after the start of treatment, with levels similar to those of healthy controls. TREM-1 expression was directly correlated with lymphocyte and erythrocyte count and indirectly correlated with spleen and liver size. Furthermore, elevated levels of TREM-1 expression were also correlated with lower serum levels of interleukin (IL)-22. Taken together, these results suggest that infection by Leishmania infantum leads to depressed TREM-1 expression on the neutrophil surface and may contribute to the inflammatory imbalance that characterizes active VL disease.

MiR-548d-3pIs Up-Regulated in Human Visceral Leishmaniasis and Suppresses Parasite Growth inMacrophages.

Visceral leishmaniasis caused by Leishmania (Leishmania) infantum in Latin America progress with hepatosplenomegaly, pancytopenia, hypergammaglobulinemia, and weight loss and maybe lethal mainly in untreated cases. miRNAs are important regulators of immune and inflammatory gene expression, but their mechanisms of action and their relationship to pathogenesis in leishmaniasis are not well understood. In the present study, we sought to quantify changes in miRNAs associated with immune and inflammatory pathways using the L. (L.) infantum promastigote infected- human monocytic THP-1 cell model and plasma from patients with visceral leishmaniasis. We identified differentially expressed miRNAs in infected THP-1 cells compared with non-infected cells using qPCR arrays. These miRNAs were submitted to in silico analysis, revealing targets within functional pathways associated with TGF-β, chemokines, glucose metabolism, inflammation, apoptosis, and cell signaling. In parallel, we identified differentially expressed miRNAs in active visceral leishmaniasis patient plasma compared with endemic healthy controls. In silico analysis of these data indicated different predicted targets within the TGF-β, TLR4, IGF-I, chemokine, and HIF1α pathways. Only a small number of miRNAs were commonly identified in these two datasets, notably with miR-548d-3p being up-regulated in both conditions. To evaluate the potential biological role of miR-548d-3p, we transiently transfected a miR-548d-3p inhibitor into L. (L.) infantum infected-THP-1 cells, finding that inhibition of miR-548d-3p enhanced parasite growth, likely mediated through reduced levels of MCP-1/CCL2 and nitric oxide production. Further work will be required to determine how miR-548d-3p plays a role in vivo and whether it serves as a potential biomarker of progressive leishmaniasis.

Evaluation of medullary cytokine expression and clinical and laboratory aspects in severe human visceral leishmaniasis.

Visceral leishmaniasis (VL) is a serious public health problem. The factors that can determine whether VL develops and progresses to severe form have not been fully identified, but a specific cellular immune response appears to play a key role. Therefore, understanding immunopathogenesis can be useful in preventing a serious clinical outcome. Bone marrow samples were collected from patients with severe VL (SVL) or non-severe VL (NSVL). Cytokine levels and parasitic load were analysed by RT-qPCR. There is a statistically significant difference in the leukocyte parameter in patients with SVL and NSVL compared with the control patients (p=.006 and p=.014, respectively). Urea, alanine transaminase and albumin parameters had a significant difference p=.036, p=.039 and p=.017, respectively, between SVL and NSVL. Although high levels of IFN-γ, IL-10, IL-6 and TNF-α were present in all groups of individuals with VL, they were not statistically associated with severity. In patients with active VL, IFN-γ and IL-10 were associated, respectively, with a reduction and increase in the parasite load, strong and significant positive association between IFN-γ and IL-10 (rho=.627 and p=.003). This study demonstrates that VL stimulates an non-dichotomized inflammatory response between Th1/Th2 and that bone marrow is an important tissue for immune regulation.

Enhancing reservoir control in the co-dynamics of HIV-VL: from mathematical modeling perspective

HIV patients are vulnerable to developing active visceral leishmaniasis (VL). To understand this complication, we studied a mathematical model for HIV and visceral leishmaniasis coinfection. In this approach, we reckoned two distinct equilibria: the disease-free and the endemic equilibria. The local and global stability of the disease-free equilibrium were thoroughly investigated. To further support the qualitative findings, we performed simulations to quantify the changes of the dynamical behavior of the full model for variation of relevant parameters. Increasing the rate of VL recovery (phi _{1}), the recovery rate for VL–HIV Co-infection (phi _{2}), removing reservoirs (c_{1}), minimizing the contact rate (beta _{h}) are important in controlling the transmission of individual and co-infection disease of VL and HIV. In conclusion, possible measures should be implemented to reduce the number of infected individuals. Therefore, we recommend that policy makers and stakeholders incorporate these measures during planing and implementation phases to control the transmission of VL–HIV co-infection.

Antibody and cytokine levels in visceral leishmaniasis patients with varied parasitemia before, during, and after treatment in patients admitted to Arba Minch General Hospital, southern Ethiopia.

Visceral leishmaniasis is a disease caused by disseminated Leishmania donovani infection which affects almost half a million people annually. Most of the patients are reported from the Indian sub-continent, Eastern Africa and Brazil. In this study, we aimed to determine the levels of antibodies and cytokines in visceral leishmaniasis patients and to examine associations of parasitemia with the clinical states of patients. A prospective study was carried out, enrolling a total of 48 active VL patients who were evaluated before, during different time points and, three months after treatment. Serum cytokine concentrations, antibody levels, parasitemia, laboratory (hematologic and biochemical) measurements, and clinical parameters were assessed. Counts of WBC and platelets, and measurements of hemoglobin (Hb) increased during treatment (P ≤ 0.05). Elevated levels of circulating IL-10, IFN-γ, and TGF-β1 were measured before treatment. The observed increase in serum IL-10 remarkably declined within 7 days after the start of treatment. Anti-leishmanial antibody index (AI) was high in all VL patients irrespective of spleen aspirate parasite grade before treatment and at different times during treatment. However, a significant (P ≤ 0.05) decrease of AI was observed 120 days post-treatment. IL-2 serum levels were below the detection limit at all sampling points. The present results suggest that IL-10, IFN-γ, and TGF-β1 can be used as markers of active visceral leishmaniasis. In addition, measuring circulating cytokines concentrations, particularly IL-10, in combination with other clinical evaluations, could be used as criteria for the cure. The observation that a high serum concentration of IFN-gamma at baseline was associated with low parasitemia deserves further investigations.

Prevalence of asymptomatic visceral leishmaniasis in human and dog, Benishangul Gumuz regional state, Western Ethiopia

BackgroundThe Benishangul-Gumuz region is an important development corridor in Ethiopia. Large-scale projects such as the Great Renaissance Dam, mining and agriculture have entailed huge environmental modifications and settlement pattern changes. There is no detailed epidemiological information on visceral leishmaniasis (VL) in the region.Materials and methodsA cross-sectional study was carried out to assess the epidemiology and risk factors associated with Leishmania infection. A leishmanin skin test (LST) was done for 1342 participants, and for 253 of them rK39 and DAT were carried out. Thirty-six dogs owned by households with LST-positive member(s) were rK39 and DAT tested. A pretested questionnaire was used to capture individual and household characteristics.ResultsOf the 89.2% (1197/1342) who availed themselves of the LST reading, 6.0% were positive. The rk39 and DAT positivity among the 253 tested were 3.2% and 5.9%, respectively. In dogs, positivity rates by rK39 and DAT were 13.9% and 5.6%, respectively. Of the household and individual risk factors, presence of a dog in the household (P = 0.005), male sex (0.003), residence woreda (0.000) and occupation (0.023) showed a strong positive association with LST positivity. Individuals who lived in households that had dogs were 2.6 times more likely to be LST positive (AOR = 2.6; 95% CI = 1.54, 4.40). Being female decreased the probability of being LST positive by 0.38 times (AOR = 0.38; 95% CI = 0.20, 0.72). People living in Guba and Kurmuk had 4.7 (AOR = 4.74, 95% CI 1.83, 12.31) and 5.9 (AOR = 5.85, 95% CI 2.27, 15.09) times more risk of being infected.ConclusionsWe demonstrated the presence of active VL transmission in the areas. Thus, we underline the need to establish the responsible vector(s) and reservoir(s) for comprehensive early containment plans to prevent potentially harmful public health and economic consequences.Graphical

Xenodiagnosis to evaluate the infectiousness of humans to sandflies in an area endemic for visceral leishmaniasis in Bihar, India: a transmission-dynamics study

SummaryBackgroundVisceral leishmaniasis, also known on the Indian subcontinent as kala-azar, is a fatal form of leishmaniasis caused by the protozoan parasite Leishmania donovani and transmitted by the bites of the vector sandfly Phlebotomus argentipes. To achieve and sustain elimination of visceral leishmaniasis, the transmission potential of individuals exposed to L donovani from across the infection spectrum needs to be elucidated. The aim of this study was to evaluate the relative infectiousness to the sandfly vector of patients with visceral leishmaniasis or post-kala-azar dermal leishmaniasis, before and after treatment, and individuals with asymptomatic infection.MethodsIn this prospective xenodiagnosis study done in Muzaffarpur district of Bihar, India, we included patients with clinically confirmed active visceral leishmaniasis or post-kala-azar dermal leishmaniasis who presented to the Kala-Azar Medical Research Center. These participants received treatment for L donovani infection. We also included asymptomatic individuals identified through a serosurvey of 17 254 people living in 26 high-transmission clusters. Eligible participants were aged 12–64 years, were HIV negative, and had clinically or serologically confirmed L donovani infection. During xenodiagnosis, the forearms or lower legs of participants were exposed to 30–35 female P argentipes sandflies for 30 min. Blood-engorged flies were held in an environmental cabinet at 28°C and 85% humidity for 60–72 h, after which flies were dissected and evaluated for L donovani infection by microscopy and quantitative PCR (qPCR). The primary endpoint was the proportion of participants with visceral leishmaniasis or post-kala-azar dermal leishmaniasis, before and after treatment, as well as asymptomatic individuals, who were infectious to sandflies, with a participant considered infectious if promastigotes were observed in one or more individual flies by microscopy, or if one or more of the pools of flies tested positive by qPCR.FindingsBetween July 12, 2016, and March 19, 2019, we recruited 287 individuals, including 77 with active visceral leishmaniasis, 26 with post-kala-azar dermal leishmaniasis, and 184 with asymptomatic infection. Of the patients with active visceral leishmaniasis, 42 (55%) were deemed infectious to sandflies by microscopy and 60 (78%) by qPCR before treatment. No patient with visceral leishmaniasis was found to be infectious by microscopy at 30 days after treatment, although six (8%) were still positive by qPCR. Before treatment, 11 (42%) of 26 patients with post-kala-azar dermal leishmaniasis were deemed infectious to sandflies by microscopy and 23 (88%) by qPCR. Of 23 patients who were available for xenodiagnosis after treatment, one remained infectious to flies by qPCR on the pooled flies, but none remained positive by microscopy. None of the 184 asymptomatic participants were infectious to sandflies.InterpretationThese findings confirm that patients with active visceral leishmaniasis and patients with post-kala-azar dermal leishmaniasis can transmit L donovani to the sandfly vector and suggest that early diagnosis and treatment could effectively remove these individuals as infection reservoirs. An important role for asymptomatic individuals in the maintenance of the transmission cycle is not supported by these data.FundingBill & Melinda Gates Foundation.

Relación entre carga parasitaria y linfocitos Th1 en biopsias de piel de perros con leishmaniosis

<div>La leishmaniosis visceral (LV) es una enfermedad sistémica crónica severa causada por Leishmania donovani o L. infantum. Varios estudios han demostrado que la susceptibilidad a LV está relacionada a altos títulos de anticuerpos circulantes y a una depresión de la inmunidad mediada por linfocitos Th1, principalmente con disminución de la producción de interferon (IFN-γ) e interleukina (IL-12). Similar a la humana, la LV canina está caracterizada por activación policlonal de células B e inmunosupresión específica. El objetivo del trabajo fue establecer la relación entre la carga parasitaria y la respuesta inmune en perros con LV. Se seleccionaron pacientes caninos con diagnóstico serológico reactivo a LV y se les extrajeron muestras para examen parasitológico directo y biopsias de piel del pabellón auricular para efectuar la técnica inmunohistoquímica con el fin de detectar linfocitos Th1 en relación con la carga parasitaria. En muestras de animales sin síntomas que registraban una elevada carga parasitaria, la cantidad de Th1 no superaba las 4 células por campo de mayor aumento, mientras que, en las muestras donde prevalecían cargas moderadas y bajas de parásitos, la cantidad de Th1 superaban las 8 células por campo de mayor aumento, pudiendo llegar hasta 10. De las muestras provenientes de animales con síntomas se observó que en aquéllas con moderada o elevada carga de parásitos, la cantidad de linfocitos Th1 no superaba las 5 células, mientras que las muestras con baja carga parasitaria superaban las 8 células por campo de mayor aumento. Se concluye que la presencia o ausencia de sintomatología en caninos con LV no depende de la carga parasitaria encontrada en las biopsias de piel. Por otra parte, se observó que la alta carga parasitaria se relaciona con inmunosupresión mediada por linfocitos Th1, mientras que en los animales con alta respuesta inmunológica, la carga parasitaria fue baja.</div>La leishmaniosis visceral (LV) es una enfermedad sistémica crónicasevera causada por Leishmania donovani o L. infantum. Varios estudios han demostradoque la susceptibilidad a LV está relacionada a altos títulos de anticuerpos circulantes y a unadepresión de la inmunidad mediada por linfocitos Th1, principalmente con disminución dela producción de interferon (IFN-γ) e interleukina (IL-12). Similar a la humana, la LV caninaestá caracterizada por activación policlonal de células B e inmunosupresión específica. Elobjetivo del trabajo fue establecer la relación entre la carga parasitaria y la respuesta inmuneen perros con LV. Se seleccionaron pacientes caninos con diagnóstico serológico reactivo aLV y se les extrajeron muestras para examen parasitológico directo y biopsias de piel del pabellónauricular para efectuar la técnica inmunohistoquímica con el fin de detectar linfocitosTh1 en relación con la carga parasitaria. En muestras de animales sin síntomas que registrabanuna elevada carga parasitaria, la cantidad de Th1 no superaba las 4 células por campode mayor aumento, mientras que, en las muestras donde prevalecían cargas moderadas ybajas de parásitos, la cantidad de Th1 superaban las 8 células por campo de mayor aumento,pudiendo llegar hasta 10. De las muestras provenientes de animales con síntomas se observóque en aquéllas con moderada o elevada carga de parásitos, la cantidad de linfocitos Th1 nosuperaba las 5 células, mientras que las muestras con baja carga parasitaria superaban las 8células por campo de mayor aumento. Se concluye que la presencia o ausencia de sintomatologíaen caninos con LV no depende de la carga parasitaria encontrada en las biopsias depiel. Por otra parte, se observó que la alta carga parasitaria se relaciona con inmunosupresiónmediada por linfocitos Th1, mientras que en los animales con alta respuesta inmunológica,la carga parasitaria fue baja.

Urine-Based Antigen (Protein) Detection Test for the Diagnosis of Visceral Leishmaniasis.

This review describes and appraises a novel protein-based antigen detection test for visceral leishmaniasis (VL). The test detects in patient’s urine six proteins from Leishmania infantum (chagasi) and Leishmania donovani, the etiological agents of VL. The gold standard test for VL is microscopic observation of the parasites in aspirates from spleen, liver, or bone marrow (and lymph node for dogs). Culture of the parasites or detection of their DNA in these aspirates are also commonly used. Serological tests are available but they cannot distinguish patients with active VL from either healthy subjects exposed to the parasites or from subjects who had a successful VL treatment. An antigen detection test based on the agglutination of anti-leishmania carbohydrates antibody coated latex beads has been described. However, the results obtained with this carbohydrate-based test have been conflicting. Using mass spectrometry, we discovered six L. infantum/L. donovani proteins excreted in the urine of VL patients and used them as markers for the development of a robust mAb-based antigen (protein) detection test. The test is assembled in a multiplexed format to simultaneously detect all six markers. Its initial clinical validation showed a sensitivity of 93% and specificity of 100% for VL diagnosis.

Conversion of asymptomatic infection to symptomatic visceral leishmaniasis: Astudy of possible immunological markers.

IntroductionPresence of asymptomatic individuals in endemic areas is common. The possible biomarkers in asymptomatic individuals once they get exposed to infection as well as following conversion to symptomatic disease are yet to be identified.We identified asymptomatic Visceral leishmaniasis (VL) infection amongst rK39+sorted direct agglutination test positive (DAT+) endemic healthy population and confirmed it by quantitative PCR(qPCR).The immunological determinants such as Adenosine deaminase (ADA), Interferon gamma (IFN-γ), Tumour Necrosis Factor alpha (TNF-α) and Interleukin 10 (IL-10)were examined to predict probable biomarkers for conversion to symptomatic VL.MethodsSample size was 5794 healthy individuals from VL endemic region. Antibody tests(DAT &rK39) were performed and later a qPCR assay was employed using kDNA specific primers and probes. Immunological biomarkers examined were ADA level by ADA–MTP kit and quantitative cytokines(IFN-γ, IL-10 and TNF-α) by ELISA.Results120 asymptomatic individuals of 308 rK39 sero-positives were DAT positive comprising of 56 with previous history and 64 with no history of VL. RT-PCR confirmed asymptomatic VL in 42 sero-positives. These were followed up through repeated qPCR and evaluation of immunological determinants. We observed10 symptomatic cases converted from a total of 42 asymptomatic individuals identified at base-line. The level of ADA, IL-10 and IFN-γ remained consistently high in asymptomatic cases and amongst these, ADA and IL-10 but not IFN-γ remained higher at the development of clinical symptoms into active VL. On the contrary, there was no significant change in the mean concentration of TNF-α at both stages of the disease.DiscussionWe surmise from our data that considerable proportion of asymptomatic cases can be a reservoir and may play a crucial role in transmission of visceral leishmaniasis in endemic areas.The data also suggests that ADA and IL-10 can serve as a potential biomarker during the conversion of asymptomatic into symptomatic VL.

Role of asymptomatic and symptomatic humans as reservoirs of visceral leishmaniasis in a Mediterranean context.

BackgroundIn the Mediterranean basin, Leishmania infantum is the causative agent of visceral leishmaniasis (VL), a zoonosis in which the dog is the primary domestic reservoir, although wildlife may have a leading role in the sylvatic cycle of the disease in some areas. Infections without disease are very frequent. There is limited information regarding the role that VL patients and asymptomatic infected individuals could be playing in the transmission of L. infantum. Xenodiagnosis of leishmaniasis has been used in this descriptive study to explore the role of symptomatic and asymptomatic infected individuals as reservoirs in a recent focus of leishmaniasis in southwestern Madrid, Spain.Methodology and main findingsAsymptomatic blood donors (n = 24), immunocompetent patients who were untreated (n = 12) or treated (n = 11) for visceral leishmaniasis (VL), and immunocompromised patients with VL (n = 3) were enrolled in the study. Their infectivity to Phlebotomus perniciosus was studied by indirect xenodiagnosis on peripheral blood samples. Quantitative polymerase chain reaction of blood samples from immunocompetent patients untreated for VL and immunocompromised untreated, treated and under secondary prophylaxis for VL was performed. Antibodies against Leishmania were studied by indirect fluorescent antibody and rK39-immunochromatographic tests. A lymphoproliferative assay with a soluble Leishmania antigen was used to screen for leishmaniasis infection in the healthy population. Sixty-two xenodiagnostic tests were carried out and 5,080 sand flies were dissected. Positive xenodiagnosis was recorded in four patients, with different sand fly infection rates: 1 immunosuppressed HIV / L. infantum coinfected asymptomatic patient, 1 immunosuppressed patient with multiple myeloma and symptomatic active VL, and 2 immunocompetent patients with untreated active VL. All blood donors were negative for both xenodiagnosis and conventional PCR.Conclusions / SignificanceThere is no consensus amongst authors on the definition of an ‘asymptomatic case’ nor on the tools for screening; we, therefore, have adopted one for the sake of clarity. Immunocompetent subjects, both infected asymptomatics and those treated for VL, are limited in number and appear to have no epidemiological relevance. The impact is limited for immunocompetent patients with untreated active VL, whilst immunosuppressed individuals undergoing immunosuppressive therapy and immunosuppressed individuals HIV / L. infantum coinfected were the most infectious towards sand flies. It is noteworthy that the HIV / L. infantum coinfected patient with asymptomatic leishmaniasis was easily infectious to sand flies for a long time, despite being under continuous prophylaxis for leishmaniasis. Accordingly, screening for latent Leishmania infection in HIV-infected patients is recommended in scenarios where transmission occurs. In addition, screening for VL in HIV-infected patients who have spent time in VL-endemic areas should also be implemented in non-endemic areas. More research is needed to better understand if some asymptomatic coinfected individuals contribute to transmission as ‘super-spreaders’.

Serum profile of IL-1β and IL-17 cytokines in patients with visceral leishmaniasis

Leishmania is an intracellular protozoan parasite, mainly infects macrophages of mammalian tissues. Inflammatory related cytokines have a crucial role in the pathogenesis of leishmaniasis. The aim of the present study was to evaluate serum concentrations of IL-1β and IL-17 in patients with active visceral leishmaniasis (VL) and control group. Serum concentrations of both IL-1β and IL-17 cytokines were assessed by ELISA in Leishmania infantum infected patients (n = 25) and healthy individuals (n = 25) from Meshkin-Shahr, northwest of Iran. Mean serum concentrations of IL-1β in the patients and control groups were 47.34 ± 23.82, and 20.49 ± 9.38, respectively, which was statistically significant (p < 0.001). Furthermore, mean IL-17 concentration in patients with VL (243.96 ± 73.46) was twice higher comparing to control group (106.38 ± 129.06) (p < 0.001). Several cytokines are involved in the regulation of immunity against VL. The present data has shown that, increased serum concentrations of IL-1β and IL-17 are present in the patients with VL. Further investigations are needed to enhance our knowledge about the regulatory role of these cytokines in leishmaniasis.

Whole Blood Stimulation Assay as a Treatment Outcome Monitoring Tool for VL Patients in Ethiopia: A Pilot Evaluation.

Visceral leishmaniasis (VL) is a lethal disease if left untreated. Current treatments produce variable rates of treatment failure and toxicity without sterile cure, rendering treatment efficacy monitoring essential. To avoid repeated invasive tissue aspirates as well as empirical treatment, there is a need for new tools that allow a less-invasive and early assessment of treatment efficacy in the field. Cross-sectional studies have suggested levels of cytokines/chemokines after whole blood stimulation as good markers of cure, but longitudinal studies are lacking. In this study, we followed 13 active VL cases in an endemic area in Ethiopia by measuring the production of IFN-γ, TNF-α, IP-10, IL-2, IL-10, MCP-1, and MIG before, during, and at the end of treatment. After 24 hours of stimulation of whole blood with soluble Leishmania antigen, we observed an early, robust, and incremental increase of IFN-γ, TNF-α, and IP-10 levels in all patients during treatment. Moreover, based on the IFN-γ levels that showed an average 13-fold increase from the time of diagnosis until the end of treatment, we could almost perfectly discriminate active from cured status. Similar concentrations and patterns were found in stimulation assays with the two main Leishmania species. The levels of IFN-γ, IP-10, or TNF-α also seemed to be inversely associated with the parasite load at baseline. Despite a 1/10 drop in concentrations, similar patterns were observed in IFN-γ and IP-10 levels when dried plasma spots were stored at 4°C for an average of 225 days. All the above evidence suggests a detectable restoration of cell-mediated immunity in VL and its association with parasite clearance. With a potential application in rural settings by means of dried plasma spots, we recommend to further explore the early diagnostic value of such assays for treatment efficacy monitoring in large cohort studies including treatment failure cases.

Cutaneous parasitism in patients with American visceral leishmaniasis in an endemic area.

INTRODUCTION: Visceral leishmaniasis (VL) represents a public health concern in several areas of the world. In the American continent, VL transmission is typically zoonotic, but humans with active VL caused by Leishmania infantum are able to infect sandflies. Thus, individuals with cutaneous parasitic infections may act as reservoirs and allow interhuman transmission. Additionally, the skin may be responsible for reactivation of the disease after therapy. This study’s objective was to evaluate cutaneous parasitism in humans with VL in an American endemic area. METHODS: A cross-sectional hospital-based study was conducted in northeast Brazil from October 2016 to April 2017. Biopsies of healthy skin for histopathology and immunohistochemistry were performed prior to treatment in all study patients. RESULTS: Twenty-two patients between the ages of five months to 78 years were included in the study. Seven patients (31.8%) tested positive for HIV. Only one patient had cutaneous parasitism, as confirmed by immunohistochemistry prior to treatment. Parasitism was not detected after treatment. CONCLUSIONS: Cutaneous parasitism in the healthy skin of humans with visceral leishmaniasis, although unusual, may be a source of infection for phlebotomine sandflies.

Transcriptome Analysis Identifies Immune Markers Related to Visceral Leishmaniasis Establishment in the Experimental Model of BALB/c Mice.

Visceral leishmaniasis (VL) caused by Leishmania donovani and L. infantum is a potentially fatal disease. To date there are no registered vaccines for disease prevention despite the fact that several vaccines are in preclinical development. Thus, new strategies are needed to improve vaccine efficacy based on a better understanding of the mechanisms mediating protective immunity and mechanisms of host immune responses subversion by immunopathogenic components of Leishmania. We found that mice vaccinated with CPA162−189-loaded p8-PLGA nanoparticles, an experimental nanovaccine, induced the differentiation of antigen-specific CD8+ T cells in spleen compared to control mice, characterized by increased dynamics of proliferation and high amounts of IFN-γ production after ex vivo re-stimulation with CPA162−189 antigen. Vaccination with CPA162−189-loaded p8-PLGA nanoparticles resulted in about 80% lower parasite load in spleen and liver at 4 weeks after challenge with L. infantum promastigotes as compared to control mice. However, 16 weeks after infection the parasite load in spleen was comparable in both mouse groups. Decreased protection levels in vaccinated mice were followed by up-regulation of the anti-inflammatory IL-10 production although at lower levels in comparison to control mice. Microarray analysis in spleen tissue at 4 weeks post challenge revealed different immune-related profiles among the two groups. Specifically, vaccinated mice were characterized by similar profile to naïve mice. On the other hand, the transcriptome of the non-vaccinated mice was dominated by increased expression of genes related to interferon type I, granulocyte chemotaxis, and immune cells suppression. This profile was significantly enriched at 16 weeks post challenge, a time-point which is relative to disease establishment, and was common for both groups, further suggesting that type I signaling and granulocyte influx has a significant role in disease establishment, pathogenesis and eventually in decreased vaccine efficacy for stimulating long-term protection. Overall, we put a spotlight on host immune networks during active VL as potential targets to improve and design more effective vaccines against disease.