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.

Evaluation of blood based quantitative PCR as a molecular diagnostic tool for post kala-azar dermal leishmaniasis (PKDL).

BACKGROUND: Post kala-azar dermal leishmaniasis (PKDL) is a consequential dermal manifestation of visceral leishmaniasis (VL), serving as a parasite reservoir. The traditional diagnostic approach, which requires an invasive skin biopsy is associated with inherent risks and necessitates skilled healthcare practitioners in sterile settings. There is a critical need for a rapid, less invasive method for Leishmania detection. The main objective of this study was to evaluate and compare the diagnostic efficacy of PCR and qPCR in detecting PKDL, utilizing both skin and blood samples and to assess the utility of blood samples for molecular diagnosis. METHODS AND RESULTS: 73 individuals exhibiting clinical symptoms of PKDL and who had tested positive for rK39 rapid diagnostic test (RDT) were enrolled in this study. For the diagnosis of PKDL, both PCR and real-time quantitative PCR (qPCR), employing SYBR Green and TaqMan assays, were performed on blood and skin matched samples. qPCR results using both TaqMan and SYBR Green assay, indicated higher parasite loads in the skin compared to blood, as evident by the Ct values. Importantly, when blood samples were used for PKDL diagnosis by qPCR, an encouraging sensitivity of 69.35% (TaqMan assay) and 79.36% (SYBR Green) were obtained, compared to 8.2% with conventional PCR. CONCLUSION: The findings of the study suggest the potential utility of blood for molecular diagnosis by qPCR, offering a less invasive alternative to skin biopsies in field setting for the early detection of parasitaemia in PKDL patients and effective management and control of the disease.

Biomarkers of disease severity in patients with visceral leishmaniasis co-infected with HIV.

Visceral leishmaniasis (VL) is caused by the protozoan Leishmania spp, transmitted by sand fly bites. VL is one of the deadliest tropical infection diseases, yet the coinfection with HIV virus drastically increases relapses, treatment failure and mortality. The concomitant action of these two pathogens leads to high cellular activation independently of the progression to AIDS. In addition, microbial translocation and bacterial infections are thought to contribute worsening the clinical picture. Identifying biomarkers associated with disease severity is of interest for clinical management of patients with VL-HIV/AIDS. Thus, we analyzed in the sera several markers including interleukins (IL-1ß, IL-6, IL-8, and IL-17), interferon-γ (IFN- γ), tumor necrosis factor (TNF), lipopolysaccharide (LPS), soluble CD14 (sCD14), macrophage migration inhibitory factor (MIF) and intestinal fatty acid-binding protein (IFABP). These markers were compared with disease severity in 24 patients with VL/HIV presenting different clinical outcomes. Disease severity was defined by the probability of death calculated using a score set system derived by the Kala-Cal® software. Probability of death ranged from 3.7% to 97.9%, with median of 28.8%. Five patients died (20%). At the univariate analysis, disease severity was correlated with TNF, IFN-γ and sCD14. LPS was positively correlated with sCD14 specifically in patients with low CD4+ count (CD4+ T-cell <200 cells/mL). Most importantly, the multivariate analysis including LPS, CD4+count and sCD14 showed that sCD14 was the only independent predictor for disease severity and death. Altogether, our results indicated that sCD14 is a powerful marker of pathogenicity and death for patients with VL-HIV/AIDS.

Biomarkers of the early response to treatment of visceral leishmaniasis: A prospective cohort study.

BACKGROUND: Early biomarkers of the response to treatment are lacking and may help to reduce mortality by the vector-borne disease visceral leishmaniasis (VL). METHODS: A prospective cohort study was conducted to investigate plasma cytokines and clinical laboratory data as biomarkers of the early response to specific treatment for VL in 36 patients. RESULTS: The mean interleukin 6 (IL-6) concentration on the 7th day was 2.3% of the pre-treatment concentration, interleukin 10 (IL-10) was 8.0%, and interleukin 8 (IL-8) was 8.2%. On the 7th day, IL-10 was below half of the pre-treatment concentration in 100.0%, IL-8 in 95.5% and IL-6 in 90.9%. The spleen and liver sizes, haemoglobin, interleukin 1 beta (IL-1ß) and tumour necrosis factor alpha (TNF-α) showed a slower recovery. Fever disappeared in 91% on the 7th day, 69.4% had a normal white cell count, and 77.8% had a normal platelet value by this time. CONCLUSIONS: The plasma cytokines IL-6, IL-10 and IL-8 were demonstrated to be excellent markers of the early response to VL treatment and if tested before the 7th day, will likely prove to be better than fever measurement.

Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection.

BACKGROUND: Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE: We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS: Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS: For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS: For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.

Higher levels of TNF and IL-4 cytokines and low miR-182 expression in visceral leishmaniasis-HIV co-infected patients.

AIMS: The aim of the present study was to assess serum cytokine and miRNA expression in visceral leishmaniasis-HIV (VL-HIV) co-infection and HIV mono-infection. METHODS AND RESULTS: We analysed 113 serum samples from HIV patients in areas endemic for leishmaniasis. The diagnosis of VL was confirmed in 65 of these 113 samples. The VL-HIV and HIV groups presented significant differences regarding haemoglobin level (P < .0001), lymphocyte count (P = .0444), white blood cell count (P = .0108), weight loss (P = .0310), HIV load (P < .0001) and CD4+ T-lymphocytes count (P = .0003). Levels of IL-6 and IL-10, IFN-γ and IL-6, IFN-γ and IL-10, TNF and IL-2 were positively correlated in VL-HIV co-infection, indicating higher serum levels of TNF and IL-4 (P < .0001). In addition, miR-182 expression was found to be significantly higher in HIV (P = .009), miR-210 exhibited no statistically significant difference between groups, and nonexpression of miR-122 was found in both groups. CONCLUSION: Together, TNF, IL-4 and miR-182 may represent circulatory biomarkers of VL-HIV co-infection.

Prospection of immunological biomarkers for characterization and monitoring of asymptomatic Leishmania (Leishmania) infantum infection.

In areas endemic for Leishmania infantum, an asymptomatic infection may be an indicator of the extent of transmission. The main goal of this study was to evaluate the applicability of measuring circulating immunological biomarkers as an alternative strategy to characterize and monitor L. infantum asymptomatic infections in combination with serological methods. To this end, 179 children from a region endemic for visceral leishmaniasis (VL), aged 1-10 years old, selected from a cross-sectional study, were identified as asymptomatic (n = 81) or uninfected (n = 98) by qPCR and/or serological tests (ELISA using L. infantum soluble antigen and rK39), and, together with serum samples of children diagnosed with VL (n = 43), were subjected to avidity tests and cytokine levels measurement. Avidity rates (AR) ranging from 41 to 70% were found in 29 children (66%) from the asymptomatic group. On the other hand, high AR (above 70%) were observed in 27 children (64%) from the VL group. Logistic Regression and Classification and Regression Tree (CART) analyses demonstrated that lower AR and IFN-γ production associated with higher IL-17A levels were hallmarks in asymptomatic L. infantum infections. Therefore, this study proposes an association of immunological biomarkers that can be used as a complementary strategy for the characterization and monitoring of asymptomatic VL infections in children living in endemic areas.

Clinical, haematological and biochemical findings in tigers infected by Leishmania infantum.

BACKGROUND: A large number of animal species are susceptible to Leishmania infantum (Kinetoplastida, Trypanosomatidae) in endemic areas, including domestic and wild felids such as tigers (Panthera tigris). Knowledge on the infection of this endangered species is still at its infancy, and therefore this study aims to identify clinical presentation and clinicopathological findings of tigers naturally infected by L. infantum. RESULTS: Tigers either L. infantum-positive (group A) or -negative (group B) were apparently healthy or presented visceral leishmaniasis unrelated conditions, except for one animal in which a large non-healing cutaneous lesion was observed. However, histological exam and immunohistochemistry carried out on the lesion excluded the presence of L. infantum amastigotes. Biochemical analysis showed that the average concentration of total proteins, globulins and haptoglobin were significantly higher (p < 0.01, p = 0.01 and p = 0.02, respectively), while the albumin/globulin ratio significantly lower (p = 0.05) in group A compared with group B. The biochemical alterations were partially confirmed by the serum protein electrophoresis results revealing a significant increase in the total protein value (p = 0.01) and hypergammaglobulinemia (p = 0.03) but an unmodified albumin/globulin ratio in group A. CONCLUSIONS: In this study tigers infected by L. infantum have shown to be mainly asymptomatic. The absence of clinical signs may lead veterinarians to overlook leishmaniasis in animals kept in captivity. Therefore, diagnostic and screening tests as serology should be part of routinely surveillance programs to be performed on tigers in zoological gardens located in endemic areas. Though only few protein-related laboratory abnormalities were recorded in infected animals, they could provide diagnostic clues for a first suspicion of L. infantum infection in tigers. Indeed, considering the high risk of zoonotic transmission in heavily frequented environment as zoos, a prompt diagnosis of L. infantum infection is of pivotal importance.

Modulation in the expression of type 1 (CR1/CD35) and type 3 (CR3/CD11b) complement receptors on leukocytes from patients with Visceral leishmaniasis.

Visceral leishmaniasis (VL) is an anthropozoonosis endemic in Brazil. We included 20 patients with confirmed diagnosis of VL and 20 healthy individuals to evaluate the expression levels of complement receptor 1 (CR1)/CD35 and CR3/CD11b on leukocytes in the peripheral blood and determine their correlation with the clinical state of patients. CR1/CD35 expression increased on CD11b+CD35+granulocytes of patients, while CR1/CD35 and CR3/CD11b expression levels increased on CD14+CD11b+CD35+ monocytes. Among patients, those with severe clinical state had higher expression of CR3/CD11b on CD14+monocytes. The count of CD19+CD35+B lymphocytes reduced in the blood samples from patients. These observed changes may indicate the modulation in CR1/CD35 and CR3/CD11b complement receptor expressionlevels on granulocyte and monocyte populations in response to Leishmania sp.

Detection of Immunoglobulin G1 Against rK39 Improves Monitoring of Treatment Outcomes in Visceral Leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL), caused by the Leishmania donovani complex, is a fatal, neglected tropical disease that is targeted for elimination in India, Nepal, and Bangladesh. Improved diagnostic tests are required for early case detection and for monitoring the outcomes of treatments. Previous investigations using Leishmania lysate antigen demonstrated that the immunoglobulin (Ig) G1 response is a potential indicator of a patient's clinical status after chemotherapy. METHODS: IgG1 or IgG enzyme-linked immunosorbent assays (ELISAs) with rK39 or lysate antigens and novel IgG1 rK39 rapid diagnostic tests (RDTs) were assessed with Indian VL serum samples from the following clinical groups: paired pre- and postchemotherapy (deemed cured); relapsed; other infectious diseases; and endemic, healthy controls. RESULTS: With paired pre- and post-treatment samples (n = 37 pairs), ELISAs with rK39- and IgG1-specific conjugates gave a far more discriminative decrease in post-treatment antibody responses when compared to IgG (P < .0001). Novel IgG1 rK39 RDTs provided strong evidence for decreased IgG1 responses in patients who had successful treatment (P < .0001). Furthermore, both IgG1 rK39 RDTs (n = 38) and ELISAs showed a highly significant difference in test outcomes between cured patients and those who relapsed (n = 23; P < .0001). RDTs were more sensitive than corresponding ELISAs. CONCLUSIONS: We present strong evidence for the use of IgG1 in monitoring treatment outcomes in VL, and the first use of an IgG1-based RDT using the rK39 antigen for the discrimination of post-treatment cure versus relapse in VL. Such an RDT may have a significant role in monitoring patients and in targeted control and elimination of this devastating disease.

Pharmacokinetics, Safety, and Efficacy of an Allometric Miltefosine Regimen for the Treatment of Visceral Leishmaniasis in Eastern African Children: An Open-label, Phase II Clinical Trial.

BACKGROUND: Convenient, safe, and effective treatments for visceral leishmaniasis in Eastern African children are lacking. Miltefosine, the only oral treatment, failed to achieve adequate efficacy, particularly in children, in whom linear dosing (2.5 mg/kg/day for 28 days) resulted in a 59% cure rate, with lower systemic exposure than in adults. METHODS: We conducted a Phase II trial in 30 children with visceral leishmaniasis, aged 4-12 years, to test whether 28 days of allometric miltefosine dosing safely achieves a higher systemic exposure than linear dosing. RESULTS: Miltefosine accumulated during treatment. Median areas under the concentration time curve from days 0-210 and plasma maximum concentration values were slightly higher than those reported previously for children on linear dosing, but not dose-proportionally. Miltefosine exposure at the start of treatment was increased, with higher median plasma concentrations on day 7 (5.88 versus 2.67 µg/mL). Concentration-time curves were less variable, avoiding the low levels of exposure observed with linear dosing. The 210-day cure rate was 90% (95% confidence interval, 73-98%), similar to that previously described in adults. There were 19 treatment-related adverse events (AEs), but none caused treatment discontinuation. There were 2 serious AEs: both were unrelated to treatment and both patients were fully recovered. CONCLUSIONS: Allometric miltefosine dosing achieved increased and less-variable exposure than linear dosing, though not reaching the expected exposure levels. The new dosing regimen safely increased the efficacy of miltefosine for Eastern African children with visceral leishmaniasis. Further development of miltefosine should adopt allometric dosing in pediatric patients. CLINICAL TRIALS REGISTRATION: NCT02431143.

Recombinant Cysteine Proteinase B from Leishmania braziliensis and Its Domains: Promising Antigens for Serodiagnosis of Cutaneous and Visceral Leishmaniasis in Dogs.

Leishmaniasis represents a group of parasitic diseases caused by a protozoan of the genus Leishmania and is widely distributed in tropical and subtropical regions. Leishmaniasis is one of the major tropical neglected diseases, with 1.5 to 2 million new cases occurring annually. Diagnosis remains a challenge despite advances in parasitological, serological, and molecular methods. Dogs are an important host for the parasite and develop both visceral and cutaneous lesions. Our goal was to contribute to the diagnosis of canine cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) using the recombinant cysteine proteinase B (F-CPB) from Leishmania braziliensis and its N- and C-terminal domains (N-CPB and C-CPB) as antigens in an enzyme-linked immunosorbent assay (ELISA). Sera from dogs from Northwest Argentina diagnosed with CL were tested by ELISA against a supernatant of L. braziliensis lysate, the F-CPB protein, and its domains. We found values of sensitivity (Se) of 90.7%, 94.4%, and 94.3% and specificity (Sp) of 95.5%, 90.9%, and 91.3% for F-CPB and its N- and C-terminal domains, respectively. In sera from dogs diagnosed with VL from Northeast Argentina, we found Se of 93.3%, 73.3%, and 66.7% and Sp of 92.3%, 76.9%, and 88.5% for F-CPB and its N- and C-terminal domains, respectively. These results support CPB as a relevant antigen for canine leishmaniasis diagnosis in its different clinical presentations. More interestingly, the amino acid sequence of CPB showed high percentages of identity in several Leishmania species, suggesting that the CPB from L. braziliensis qualifies as a good antigen for the diagnosis of leishmaniasis caused by different species.

Canine visceral leishmaniasis: Detection of Leishmania spp. genome in peripheral blood of seropositive dogs by real-time polymerase chain reaction (rt-PCR).

Visceral leishmaniasis (VL) is a zoonosis caused by the protozoa of the genus Leishmania. Among the species, L. infantum and/or L. infantum (chagasi) are the most important species affecting the Americas. Domestic dogs are the main reservoir of the parasite and participate effectively in the parasite' transmission cycle. The Canine Visceral Leishmaniasis Control Program (PCLV) adopted in Brazil present as strategies the vector control, health education and serological diagnosis of CVL in dogs followed by culling of the seropositive ones. The resolution to eliminate seropositive dogs by euthanasia, when necessary, are the most controversial and least accepted by society. The diagnostic methods for canine visceral leishmaniasis, currently indicated and approved in Brazil by the Ministry of Health from Brazil are the Dual Path Platform (DPP)® as a screening test and the Enzyme immunoassay test (ELISA®). This study aimed to verify the presence of Leishmania spp. DNA in peripheral blood samples of dogs presenting positive serological results byDPP® and ELISA® tests,throughreal-time polymerase chain reaction (rt-PCR), using the pair of primers 150-152 already described. For this purpose, were collected blood samples from 185 seropositive dogs among them, 41 (22%) exhibited some clinical signal of disease, whereas 144 (78%) was asymptomatic. The animals were also analyzed according to gender, race and hair size. According to the results of rt-PCR, it was observed that among the185 seropositive dogs analyzed, only 132 (71%) presented positive results for CVL and 53 (29%) presented negative results. From this, 41/41 symptomatic dogs were positive (100%), while among the asymptomatic dogs, 91/144 were positive (63, 2%) and 53/144 were negative (36, 8%). Concerning the hair size of seropositive dogs, we found that 41 (22%) had long hair, while 144 (78%) had short hair. No statistical significance occurred between the results of rt-PCR, ELISA and DPP tests and the profile of the animals (gender, size of the dogs and hair size), probably due to the small number of samples and the sampling differences of each profile. But statistical significance occurred between the results of rt-PCR and the clinical evaluation, since the rt-PCR was positive in all symptomatic dogs. Thus, through these results, we reached at the following question, which may contribute to an important current debate: the dogs presenting CVL seropositive diagnosis confirmed by tests distributed by the Ministry of Health were in reality ill or were they seropositive by living in an endemic area of the disease? Would these asymptomatic seropositive dogs spread the disease to the inhabitants even presenting a low parasite charge circulating in the blood.

Verification and monitoring of visceral leishmaniasis in hamsters caused by Leishmania infantum, using non-invasive approaches involving ultrasound imaging and blood gases.

Hamsters are a suitable experimental model for visceral leishmaniasis (VL) because they mimic the features of the human disease. However, the infection after inoculation can only be verified after sacrifice of the animal or several months following infection, when obvious signs of the disease appear, compromising animal welfare in both cases. Unlike other studies, the present work used an inoculum of 5 × 108 promastigotes to induce Leishmania infantum infection, which are easier to produce than amastigotes, in in vitro culture. The infection in hamsters was detected using non-invasive methods such as ultrasound imaging (USG) and blood gases, in addition to alterations in hematological parameters and weight loss. USG imaging identified changes in the size and echogenicity of the spleen, liver, and kidney as early as week 9 (W9) after experimental inoculation. However, blood gases, specially lactate, was increased in response to the infection, with statistically significant differences between W9 and W0 (before infection) (p < 0.0001). The conventional hematological parameters showed progressive pancytopenia and weight loss of 15% and 10% in infected males and females respectively, at W9 versus W0 (p < 0.0001). Histological changes in the liver, kidney, and spleen correlated with changes detected by USG imaging and the number of parasites increased proportionately to the progression of infection, being higher at W24. In conclusion, USG imaging, lactate levels, hematocrit and hemoglobin parameters, along with weight loss allowed early detection of infection, which was then confirmed by the identification and quantification of parasites in the blood, liver, and spleen by qRT-PCR. In contrast, blood chemistry was not a useful tool in the early detection of VL infection because it did not correlate with alterations evident in other techniques. The use of non-invasive tools eliminates the need for animal sacrifice to confirm infection, thus reducing the number of animals required for a given study and eliminating the need to wait until the appearance of severe signs of infection, which affect animal welfare. These tools are therefore advantageous for use in preclinical studies, for studying pathogenesis as also for vaccine and drug development.

Dermatological Implications of Galectin-3 in Circulation: An Evaluation From the Perspective of Patients With Differential Manifestations of Post-Kala-Azar Dermal Leishmaniasis.

Galectin-3, a ß-galactoside-binding lectin, has been implicated in vast repertoire of inflammatory and immunomodulatory processes including skin diseases. However, galectin-3 has not been comprehensively studied in infectious diseases. This study emphasizes on fascinating aspects of galectin-3 expression in dermal infection by studying post-kala-azar dermal leishmaniasis (PKDL), an intracellular infection caused by Leishmania donovani. Indian PKDL is a well-recognized parasitic dermatosis, with a high risk of anthroponotic transmission of L. donovani in causing leishmaniasis. This study aims to investigate the levels of galectin-3 and galectin-3-binding site expression in circulation of different forms of Indian patients with PKDL. Thirty-seven confirmed untreated PKDL patients, comprising 20 polymorphic and 17 macular PKDL manifestations, were evaluated for the levels of sera galectin-3 with respect to 28 age- and sex-matched healthy controls from endemic areas. Result shows a significant increment (P < 0.001) in circulatory galectin-3 levels in PKDL variants as compared to healthy controls. In addition, there were heightened levels of galectin-3 and galectin-3-binding sites on cellular infiltrates on lesional sites. Furthermore, there was a positive correlation between frequencies of mononuclear cells and galectin-3 during microcirculation in lesions. Data were well corroborated with positive correlation of IL-10 and IFN-γ with sera galectin-3 levels. Moreover, flow cytometry demonstrated the enhanced expression levels of the galectin-3-binding site in circulation in patients with PKDL as compared to healthy controls. Taken together, elevated levels of galectin-3 reflect its involvement in PKDL pathogenesis.

Serological and molecular investigation of selected parasitic pathogens in European brown hare (Lepus europaeus) in Greece: inferring the ecological niche of Toxoplasma gondii and Leishmania infantum in hares.

The occurrence of infection or exposure to Toxoplasma gondii, Neospora caninum, and Leishmania infantum was investigated in European brown hares (Lepus europaeus, EBH) hunter-harvested over two consecutive hunting seasons in northern and central Greece. Geographical information system was used along with the ecological niche model to define the geographical distribution of seropositive hares relative to environmental parameters and to identify high-risk areas for hare exposure. Molecular analysis showed that 3.8% and 9.6% of the examined hares were infected with N. caninum and L. infantum, respectively, while, 5.7%, 0.95%, and 12.4% of the hares tested positive for the presence of antibodies against T. gondii, N. caninum, and L. infantum respectively. None of the examined hares was polymerase chain reaction-positive for T. gondii. Mixed exposure against both T. gondii and L. infantum was found in 2.9% of the hares examined. Rainfall indices and land uses significantly influenced the exposure of hares to T. gondii and L. infantum. This is the first molecular and serological survey of protozoan pathogens in EBH in Greece. Furthermore, we report the environmental parameters related to hare seropositivity and present a risk map for hare exposure to T. gondii and L. infantum in northern and central Greece. The ecological niches of T. gondii and L. infantum in the hares presented herein could be applied to other regions with similar environmental and climatic conditions.

Leishmania donovani reduces the levels of retinoic acid-synthesizing enzymes in infected macrophages and favoring its own survival.

People suffering from malnutrition become susceptible to the infection like Leishmania sp., as it results in a compromised immune response. Retinoic acid (RA), an important constituent of nutrition, shows an immune-modulatory activity. However, its role in the containment of infection is not yet ascertained, particularly in case of visceral leishmaniasis (VL). VL patients (n = 10) and healthy endemic controls (n = 9) were recruited to measure the serum levels of RA. An in vitro model of Leishmania infection using the murine mφ cell line J774.1 was used to investigate the RA-synthesizing enzymes (RALDH-1 and RALDH-2). Parasite loads among infected mφ were measured by quantitative expression of kDNA in the presence of an inhibitor of the RALDH-2 enzyme. We found a significant decrease in the serum levels of RA in VL cases. Importantly, we observed decreased levels of RALDH-1 and RALDH-2 among L. donovani-infected mφ along with simultaneous decrease as well as increase in the Th-1 and Th-2-associated factors, respectively. Furthermore, the pretreatment of mφ with an RALDH-2 inhibitor improved parasite in vitro infection. Our findings show impaired RA pathway among infected mφ and indicate that an intact RA pathway is critical for anti-Leishmania immune response. Graphical abstract ᅟ.

Prevalence of asymptomatic Leishmania infection and associated risk factors, after an outbreak in the south-western Madrid region, Spain, 2015.

BackgroundA large outbreak of leishmaniasis with 758 cutaneous and visceral leishmaniasis cases occurred in 2009 in Fuenlabrada, in the south-west of the Madrid region of Spain.AimWe aimed to determine the prevalence of asymptomatic Leishmania infection after this outbreak, and its associated risk factors.MethodsA cross-sectional study of 804 healthy individuals living in Fuenlabrada who had no history of leishmaniasis, was conducted between January and July 2015. Asymptomatic infections were sought by either a combination of PCR, immunofluorescent antibody titre, and direct agglutination tests, or by whole blood stimulation assay (WBA) with interleukin-2 (IL-2) quantification.ResultsUsing the first approach, prevalence of asymptomatic individuals was 1.1% (9/804), while the second returned a value of 20.7% (143/804). Older age, being male, proximity to the park where the focus of infection was identified, and living in a detached house, were all strongly associated with the prevalence of asymptomatic infection.ConclusionsThe true number of infected individuals may be underestimated if only serological methods are used. The combination of WBA with IL-2 quantification may allow to better determine the prevalence of asymptomatic Leishmania infection, which would be useful in establishing control measures and in quantifying their impact. In our study, the use of WBA with IL-2 quantification also helped establish the risk factors that influence exposure to and infection by Leishmania.

[Pancytopenia of unknown origin in a 52-year-old patient]. / Unklare Panzytopenie bei einem 52-jährigen Patienten.

A 52-year-old patient developed pancytopenia of unknown origin 1.5 years after allogeneic stem cell transplantation. The bone marrow aspirate showed visceral leishmaniasis (VL). Although VL is distributed world-wide, the incidence in patients after allogeneic stem cell transplantation is rare.

Visceral leishmaniasis-associated nephropathy in hospitalised Brazilian patients: new insights based on kidney injury biomarkers.

OBJECTIVE: To evaluate the usefulness of early acute kidney injury (AKI) biomarkers in clinical management of visceral leishmaniasis. METHODS: Prospective study with 50 hospitalised VL patients. AKI biomarkers, that is, serum and urinary neutrophil gelatinase-associated lipocalin (sNGAL, uNGAL, respectively), urinary kidney injury molecule-1 (uKIM-1) and urinary monocyte chemotactic protein-1 (uMCP-1), were quantified by immunoassay (ELISA). Also, interferon-gamma (INF-y) and C-reactive protein (CRP) were evaluated as inflammatory biomarkers possibly related to VL severity. RESULTS: VL patients had hyponatremia, hypoalbuminemia, hypergammaglobulinemia, haematologic and hepatic disorders. AKI was found in 46%, and one death (2%) occurred. The AKI group had significant longer hospital stay, lower levels of IFN-y and higher levels of CRP, more clinical renal abnormalities and higher levels of sNGAL, uNGAL, uKIM-1 and uMCP-1. Overall, sNGAL, uKIM-1 and uMCP-1 showed correlations with important clinical renal abnormalities, such as proteinuria, albuminuria, serum creatinine and glomerular filtration rate using adjusted correlations with CRP and IFN-y. Only sNGAL showed an early association with AKI development (OR = 2.78, 95% CI = 1.429-5.428, per each increase of 50 ng/ml), even after adjusting for clinical signals of VL severity and for immune biomarkers. Moreover, sNGAL showed a better performance in predicting AKI development (AUC-ROC = 0.81, 95% CI = 0.69-0.93; cut-off = 154 ng/ml, sensitivity = 82.6%, specificity = 74.1%, P < 0.001). CONCLUSIONS: Visceral leishmaniasis-associated nephropathy showed important proximal tubular injury and glomerular inflammation. Serum NGAL showed an early association with VL-associated nephropathy and may be used to improve clinical management strategies and decrease morbimortality in VL patients.