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BACKGROUND: The host cellular immune response associated with two treatments for post-kala-azar dermal leishmaniasis (PKDL) - paromomycin plus miltefosine (Arm 1), and liposomal amphotericin B plus miltefosine (Arm 2) - was examined in Sudanese patients before treatment (D0), at the end of treatment (D42), and during the post-treatment period (D180). METHODS: Whole blood samples were stimulated with soluble Leishmania antigen for 24 h (whole blood assay [WBA]) and the concentrations of Th1/Th2/Th17-associated cytokines, IP-10, PDL-1 and granzyme B were determined. RESULTS: The Arm 1 treatment (98.2% cure rate) induced a Th1/Th2/Th17 response, while the Arm 2 treatment (80% cure rate) induced a Th1/Th2 response. Five Arm 2 patients relapsed and showed lower IFN-γ, TNF and IL-1ß concentrations at D0 than non-relapsers in this Arm. In patients with low-IFN-γ-production at D0, Arm 1 treatment led to a better host immune response and clinical outcome than Arm 2 treatment. CONCLUSIONS: A Th1/Th2/Th17 response was associated with a higher cure rate. Patients with low IFN-γ, TNF and IL-1ß before treatment are more likely to relapse if they undergo Arm 2-type treatment. Determining IFN-γ, TNF and IL-10 levels prior to treatment could help predict patients at higher risk of relapse/recovery from PKDL. TRIAL REGISTRATION: ClinicalTrials.gov NCT03399955, Registered 17 January 2018, https://clinicaltrials.gov/study/ NCT03399955.
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BACKGROUND: The lack of standardized protocols for isolating extracellular vesicles (EVs), especially from biobank-stored blood plasma, translates to limitations for the study of new biomarkers. This study examines whether a combination of current isolation methods could enhance the specificity and purity of isolated EVs for diagnosis and personalized medicine purposes. RESULTS: EVs were isolated from healthy human plasma stored for one year by ultracentrifugation (UC), size exclusion chromatography (SEC), or SEC and UC combined (SEC + UC). The EV isolates were then characterized by transmission electron microscopy imaging, nanoparticle tracking analysis (NTA) and western blotting. Proteomic procedures were used to analyze protein contents. The presence of EV markers in all isolates was confirmed by western blotting yet this analysis revealed higher albumin expression in EVs-UC, suggesting plasma protein contamination. Proteomic analysis identified 542 proteins, SEC + UC yielding the most complex proteome at 364 proteins. Through gene ontology enrichment, we observed differences in the cellular components of EVs and plasma in that SEC + UC isolates featured higher proportions of EV proteins than those derived from the other two methods. Analysis of proteins unique to each isolation method served to identify 181 unique proteins for the combined approach, including those normally appearing in low concentrations in plasma. This indicates that with this combined method, it is possible to detect less abundant plasma proteins by proteomics in the resultant isolates. CONCLUSIONS: Our findings reveal that the SEC + UC approach yields highly pure and diverse EVs suitable for comprehensive proteomic analysis with applications for the detection of new biomarkers in biobank-stored plasma samples.
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BACKGROUND: Diagnosis of visceral leishmaniasis (VL) in resource-limited endemic regions is currently based on serological testing with rK39 immunochromatographic tests (ICTs). However, rK39 ICT frequently has suboptimal diagnostic accuracy. Furthermore, treatment monitoring and detection of VL relapses is reliant on insensitive and highly invasive tissue aspirate microscopy. Miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative and user-friendly molecular tool which does not require DNA extraction and uses a lateral flow strip for result read-out. This assay could be an interesting candidate for more reliable VL diagnosis and safer test of cure at the point of care. METHODOLOGY/PRINCIPLE FINDINGS: The performance of mini-dbPCR-NALFIA for diagnosis of VL in blood was assessed in a laboratory evaluation and compared with the accuracy of rK39 ICTs Kalazar Detect in Spain and IT LEISH in East Africa. Limit of detection of mini-dbPCR-NALFIA was 650 and 500 parasites per mL of blood for Leishmania donovani and Leishmania infantum, respectively. In 146 blood samples from VL-suspected patients from Spain, mini-dbPCR-NALFIA had a sensitivity of 95.8% and specificity 97.2%, while Kalazar Detect had a sensitivity of 71.2% and specificity of 94.5%, compared to a nested PCR reference. For a sample set from 58 VL patients, 10 malaria patients and 68 healthy controls from Ethiopia and Kenya, mini-dbPCR-NALFIA had a pooled sensitivity of 87.9% and pooled specificity of 100% using quantitative PCR as reference standard. IT LEISH sensitivity and specificity in the East African samples were 87.9% and 97.4%, respectively. CONCLUSIONS/SIGNIFICANCE: Mini-dbPCR-NALFIA is a promising tool for simplified molecular diagnosis of VL and follow-up of treated patients in blood samples. Future studies should evaluate its use in endemic, resource-limited settings, where mini-dbPCR-NALFIA may provide an accurate and versatile alternative to rK39 ICTs and aspirate microscopy.
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Leishmania donovani , Leishmaniose Visceral , Sensibilidade e Especificidade , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/parasitologia , Humanos , Leishmania donovani/genética , Leishmania donovani/isolamento & purificação , Imunoensaio/métodos , Leishmania infantum/genética , Leishmania infantum/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Espanha , Técnicas de Diagnóstico Molecular/métodos , Feminino , Masculino , Adulto , Adolescente , Criança , Adulto Jovem , Pessoa de Meia-Idade , África Oriental , DNA de Protozoário/genética , DNA de Protozoário/sangue , Pré-EscolarRESUMO
BACKGROUND: Radicular cyst is a lesion of odontogenic origin that arises from epithelial remains due to periapical periodontitis caused by inflammatory reactions generated at the apex of affected teeth with infected or necrotic pulps. The therapeutic management of radicular cysts is controversial. There is only one case report of enucleation of a radicular cyst managed with microsurgery and apicoectomy, but without the use of the guided tissue regeneration (GTR) technique in the same surgical procedure. The present clinical case describes the management of a radicular cyst with microsurgical approach, performance of an apicoectomy of the tooth associated with the entity, application of GTR technique, use of a resorbable membrane of type I bovine collagen, and bovine xenograft. CASE SUMMARY: A 68-year-old patient presented with a radicular cyst from an upper lateral incisor. The microsurgical management used was aimed at enucleating the chemical membrane, performing apicoectomy of the tooth along with careful and precise retrograde filling, and implementing GTR technique using a resorbable collagen membrane and bovine xenograft. The diagnosis of radicular cyst was confirmed using histopathological analysis. The patient underwent follow-up evaluations at 10 and 30 d postoperatively. At 4 months postoperative evaluation, she remained asymptomatic, and radiographs showed significant periapical healing with adequate bone formation. CONCLUSION: These results suggest that microsurgical management using the GTR technique with collagen membrane and xenograft, contributes to bone regeneration.
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BACKGROUND: Visceral leishmaniasis (VL) resolution depends on a wide range of factors, including the instauration of an effective treatment coupled to a functional host immune system. Patients with a depressed immune system, like the ones receiving methotrexate (MTX), are at higher risk of developing VL and refusing antileishmanial drugs. Moreover, the alarmingly growing levels of antimicrobial resistance, especially in endemic areas, contribute to the increasing the burden of this complex zoonotic disease. PRINCIPAL FINDINGS: To understand the potential links between immunosuppressants and antileishmanial drugs, we have studied the interaction of antimony (Sb) and MTX in a Leishmania infantum reference strain (LiWT) and in two L. infantum clinical strains (LiFS-A and LiFS-B) naturally circulating in non-treated VL dogs in Spain. The LiFS-A strain was isolated before Sb treatment in a case that responded positively to the treatment, while the LiFS-B strain was recovered from a dog before Sb treatment, with the dog later relapsing after the treatment. Our results show that, exposure to Sb or MTX leads to an increase in the production of reactive oxygen species (ROS) in LiWT which correlates with a sensitive phenotype against both drugs in promastigotes and intracellular amastigotes. LiFS-A was sensitive against Sb but resistant against MTX, displaying high levels of protection against ROS when exposed to MTX. LiFS-B was resistant to both drugs. Evaluation of the melting proteomes of the two LiFS, in the presence and absence of Sb and MTX, showed a differential enrichment of direct and indirect targets for both drugs, including common and unique pathways. CONCLUSION: Our results show the potential selection of Sb-MTX cross-resistant parasites in the field, pointing to the possibility to undermine antileishmanial treatment of those patients being treated with immunosuppressant drugs in Leishmania endemic areas.
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Antiprotozoários , Leishmania infantum , Leishmaniose Visceral , Humanos , Animais , Cães , Metotrexato/farmacologia , Metotrexato/uso terapêutico , Antimônio/farmacologia , Antimônio/uso terapêutico , Espécies Reativas de Oxigênio , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/veterinária , Resistência a MedicamentosRESUMO
OBJECTIVES: In immunocompromised patients, asymptomatic Leishmania infection can reactivate, and evolve to severe disease. To date, no test is considered the gold standard for the identification of asymptomatic Leishmania infection. A combination of methods was employed to screen for Leishmania infection in patients undergoing kidney transplant (KT). METHODS: We employed polymerase chain reaction for the detection of parasitic DNA in peripheral blood, Western blot to identify serum immunoglobulin G and whole blood assay to detect cytokines/chemokines after stimulation of whole blood with parasitic antigen. RESULTS: One-hundred twenty patients residing in Italy were included in the study at the time of KT. Each patient that tested positive to at least one test was considered as Leishmania positive. Fifty out of 120 patients (42%) tested positive for one or more tests. The detection of specific cell-mediated response (32/111, 29%) was the most common marker of Leishmania infection, followed by a positive serology (24/120, 20%). Four patients (3%) harbored parasitic DNA in the blood. CONCLUSION: Our findings underline the high prevalence of asymptomatic Leishmania infection in patients undergoing KT in Italy, who are potentially at-risk for parasite reactivation and can benefit from an increased vigilance. Understanding the clinical relevance of these findings deserves further studies.
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Transplante de Rim , Leishmania infantum , Leishmania , Leishmaniose Visceral , Leishmaniose , Humanos , Leishmania/genética , Leishmaniose Visceral/diagnóstico , Transplante de Rim/efeitos adversos , Leishmaniose/diagnóstico , Leishmaniose/epidemiologia , Infecções Assintomáticas/epidemiologia , DNARESUMO
Background: Immunosuppression is a major risk factor for the development of visceral leishmaniasis (VL). The number of patients receiving immunosuppressant drugs such as TNF antagonist (anti-TNF) and methotrexate (MTX) is increasing. In these patients, VL is more severe, their response to treatment poorer, and they are at higher risk of relapse, a consequence (largely) of the poor and inappropriate immune response they develop. Objectives: To examine the effect of immunosuppressive treatment on the host immune response and thus gain insight into the reduced efficacy of pentavalent antimonials in these patients. Experiments were performed using BALB/c mice immunosuppressed with anti-TNF or MTX, infected with Leishmania infantum promastigotes, and then treated with Glucantime® at clinical doses. Results: Immunosuppression with both agents impeded parasite elimination from the spleen and bone marrow. Low pro-inflammatory cytokine production by CD4+ and CD8+ T cells was detected, along with an increase in PD-1 and IL-10 expression by B and T cells in the immunosuppressed groups after treatment. Conclusion: The immunosuppressed mice were unable to develop specific cellular immunity to the parasite, perhaps explaining the greater risk of VL relapse seen in pharmacologically immunosuppressed human patients.
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Leishmania infantum , Leishmaniose Visceral , Leishmaniose , Parasitos , Humanos , Animais , Camundongos , Antimoniato de Meglumina/uso terapêutico , Linfócitos T CD8-Positivos , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Inibidores do Fator de Necrose Tumoral , Modelos Animais de Doenças , Imunidade Celular , RecidivaRESUMO
Acute febrile illnesses are still a major cause of mortality and morbidity globally, particularly in low to middle income countries. The aim of this study was to determine any possible metabolic commonalities of patients infected with disparate pathogens that cause fever. Three liquid chromatography-mass spectrometry (LC-MS) datasets investigating the metabolic effects of malaria, leishmaniasis and Zika virus infection were used. The retention time (RT) drift between the datasets was determined using landmarks obtained from the internal standards generally used in the quality control of the LC-MS experiments. Fitted Gaussian Process models (GPs) were used to perform a high level correction of the RT drift between the experiments, which was followed by standard peakset alignment between the samples with corrected RTs of the three LC-MS datasets. Statistical analysis, annotation and pathway analysis of the integrated peaksets were subsequently performed. Metabolic dysregulation patterns common across the datasets were identified, with kynurenine pathway being the most affected pathway between all three fever-associated datasets.
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Infecção por Zika virus , Zika virus , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Algoritmos , Metabolômica/métodosRESUMO
BACKGROUND: Most people infected with Leishmania remain asymptomatic, which is a common element that may promote the resurgence of clinically evident leishmaniasis in individuals with impaired cell-mediated immune responses. Unfortunately, there is no universally accepted assay to identify asymptomatic infection. This cross-sectional study focuses on the employment of three methods targeting different features of the parasitic infection to be used in combination for the screening of latent leishmaniasis in a newly endemic area of northeastern Italy. METHODOLOGY/PRINCIPAL FINDINGS: The selected methods included highly sensitive Real-Time PCR for detection of parasitic kinetoplast (k)DNA in peripheral blood, Western Blot (WB) for detection of specific IgG, and Whole Blood stimulation Assay (WBA) to evaluate the anti-leishmanial T-cell response by quantifying the production of IL-2 after stimulation of patients' blood with Leishmania specific antigens. Among 145 individuals living in a municipality of the Bologna province, northeastern Italy, recruited and screened for Leishmania infection, 23 subjects tested positive (15.9%) to one or more tests. Positive serology was the most common marker of latent leishmaniasis (15/145, 10%), followed by the detection of specific cell-mediated response (12/145, 8%), while only few individuals (6/145, 4%) harbored parasitic DNA in the blood. CONCLUSIONS/SIGNIFICANCE: Combining different tests substantially increased the yield of positivity in detecting latent Leishmania infection. The test combination that we employed in this study appears to be effective to accurately identify latent leishmaniasis in an endemic area.
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Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Leishmaniose , Animais , Estudos Transversais , Doenças do Cão/epidemiologia , Cães , Humanos , Leishmania infantum/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologiaRESUMO
OBJECTIVE: Sensitive and less laborious assays are needed to detect asymptomatic Leishmania among solid organ transplant (SOT) recipients. Using SLA-stimulated plasma from SOT recipients living where an outbreak of Leishmania infantum occurred, we examined potential biomarkers to identify asymptomatic Leishmania infections. METHODS: Concentrations of cytokines/chemokines in plasma from whole blood stimulated with specific Leishmania antigen (SLA) were compared against infection status as determined by a currently used cell proliferation assay. RESULTS: Twenty-six percent (13/50) of the SOT recipients had a cell proliferation assay (CPA) indicating asymptomatic infection, and showed higher processed plasma C-X-C motif chemokine ligand 10 (CXCL10 or IP-10) concentrations than did non-infected subjects (median 2272.0 pg/ml [IQR 1570-2772] vs. 18.2 pg/ml [IQR 1-150.1]; p<0.0001). CXCL10 showed a sensitivity of 93% and a specificity of 95% compared to CPA. In addition, we demonstrated that the number of asymptomatic infections detected using CXCL10, decreased with distance from a park at the center of the mentioned outbreak. CONCLUSION: CXCL10 in plasma from SLA-stimulated blood could be a robust biomarker of asymptomatic L. infantum infection in solid organ transplant recipients.
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Leishmania infantum , Leishmaniose Visceral , Transplante de Órgãos , Infecções Assintomáticas , Biomarcadores , Quimiocina CXCL10 , Humanos , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/epidemiologia , Ligantes , TransplantadosRESUMO
Visceral leishmaniasis (VL) is the most severe clinical form of leishmaniasis, fatal if untreated. Vaccination is the most cost-effective approach to disease control; however, to date, no vaccines against human VL have been made available. This work examines the efficacy of a novel vaccine consisting of the Leishmania membrane protein KMP11, LEISH-F3+ (a recombinant fusion protein, composed of epitopes of the parasite proteins nucleoside hydrolase, sterol-24-c-methyltransferase, and cysteine protease B), and the sand fly salivary protein LJL143, in two dose ratios. The inclusion of the TLR4 agonist GLA-SE as an adjuvant, and the use of virosomes (VS) as a delivery system, are also examined. In a hamster model of VL, the vaccine elicited antigen-specific immune responses prior to infection with Leishmania infantum. Of note, the responses were greater when higher doses of KMP11 and LEISH-F3+ proteins were administered along with the GLA-SE adjuvant and/or when delivered within VS. Remarkably, hamsters immunized with the complete combination (i.e., all antigens in VS + GLA-SE) showed significantly lower parasite burdens in the spleen compared to those in control animals. This protection was underpinned by a more intense, specific humoral response against the KMP11, LEISH-F3+, and LJL143 antigens in vaccinated animals, but a significantly less intense antibody response to the pool of soluble Leishmania antigens (SLA). Overall, these results indicate that this innovative vaccine formulation confers protection against L. infantum infection, supporting the advancement of the vaccine formulation into process development and manufacturing and the conduction of toxicity studies towards future phase I human clinical trials.
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Visceral leishmaniasis is associated with a variety of hematological abnormalities. In this study, we correlated the hematological changes in the peripheral blood of dogs naturally infected with Leishmania infantum (L. infantum) with the distribution of cell lineages and cytokine gene expression patterns in the bone marrow. Samples from 63 naturally semidomiciled dogs living in an endemic area of visceral leishmaniasis were analyzed. L. infantum infection was detected in 50 dogs (79.3%). Among those, 18 (32%) had positive splenic cultures and showed more clinical signs. They also had lower red blood cell counts and leukocytosis with an increased number of neutrophils and monocytes in peripheral blood compared to dogs negative to this test. L. infantum DNA was detected in the bone marrow of 8/14 dogs with positive splenic culture. Dogs with L. infantum infection in the bone marrow presented with histiocytosis (p = 0.0046), fewer erythroid cell clusters (p = 0.0127) and increased gene expression levels of IFN-γ (p = 0.0015) and TNF (p = 0.0091). The data shown herein suggest that inflammatory and cytokine gene expression changes in bone marrow may contribute to the peripheral blood hematological changes observed in visceral leishmaniasis.
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Visceral leishmaniasis (VL) in patients receiving immunosuppressant drugs for autoimmune disease has been on the rise. It is important-but difficult-to know when cure has been achieved in these patients since the withdrawal of immunosuppressants during antileishmania treatment is commonly required, and there is a risk of relapse when immunosuppression is restored. The prevalence of asymptomatic infection among those immunosuppressed for autoimmune disease is also uncertain. The present work describes how cytokine release assays can be used to confirm the cure of VL, and to determine the prevalence of asymptomatic infection, in such patients. After collection of blood from volunteers (n = 108), SLA-stimulation of peripheral blood mononuclear cell cultures and of whole blood was found to induce the production of different combinations of cytokines that served to confirm recovery from VL, and asymptomatic Leishmania infection. Indeed, cure was confirmed in 14 patients, all of whom showed a specific Th1 immune response against Leishmania, and the prevalence of asymptomatic infection was determined as 21.27%. Cytokine profiles could be used to manage VL in patients with autoimmune disease, and to identify and better protect those with asymptomatic infection who are at risk of developing this disease.
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Técnicas de Laboratório Clínico/métodos , Citocinas/metabolismo , Monitoramento de Medicamentos/métodos , Leishmaniose/diagnóstico , Leucócitos Mononucleares/imunologia , Adulto , Idoso , Anticorpos Antiprotozoários/sangue , Infecções Assintomáticas , Doenças Autoimunes/tratamento farmacológico , Humanos , Imunossupressores/uso terapêutico , Leishmaniose/epidemiologia , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Cutaneous leishmaniasis (CL) is the most common type of leishmaniasis, a neglected tropical disease caused by parasites of the genus Leishmania. In Ghana, some studies in the Volta region have detected Leishmania parasites among persons with skin ulcers. METHODOLOGY/PRINCIPAL FINDINGS: Using a cross-sectional study design, the prevalence of CL in three communities of the Oti Region of Ghana was investigated. Demographic and epidemiological data were obtained by a structured interviewer administered questionnaire. A total of 426 (12.4%) out of 3,440 participants screened had at least one skin ulcer. Of 595 skin ulcers sampled and tested by PCR for Leishmania infection, 150 (25.2%) ulcers from 136 individuals tested positive, accounting for an overall CL prevalence of 31.9% among persons with skin ulcers. Individual community CL prevalence of 23.2%, 29.8%, and 36.8% was observed in Ashiabre, Keri, and Sibi Hilltop respectively among persons with skin ulcers. CONCLUSIONS/SIGNIFICANCE: Confirmation of CL in the study area suggests an active cycle of transmission of Leishmania infection. The observation of skin ulcers which tested negative to Leishmania infection suggests a need to test for additional causes of skin ulcers such as Treponema pallidum pertenue and Mycobacterium ulcerans in the study area.
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Leishmania/isolamento & purificação , Leishmaniose Cutânea/epidemiologia , Úlcera Cutânea/epidemiologia , Adolescente , Adulto , Criança , Pré-Escolar , Estudos Transversais , Feminino , Gana/epidemiologia , Humanos , Leishmania/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prevalência , Úlcera Cutânea/parasitologia , Inquéritos e QuestionáriosRESUMO
Loop-mediated isothermal amplification allows the rapid, sensitive and specific amplification of DNA without complex and expensive equipment. We compared the diagnostic performance of Loopamp™ Leishmania Detection Kit (Eiken Chemical Co., Ltd., Tokyo, Japan) with conventional and real-time polymerase chain reaction (PCR) for human cutaneous and visceral leishmaniasis caused by L. infantum. A total of 230 DNA samples from cutaneous (CL) and visceral (VL) leishmaniasis cases and controls from Spain, characterized by Leishmania nested PCR (LnPCR) were tested by: (i) the Loopamp™ Leishmania Detection Kit (Loopamp), run on Genie III real-time fluorimeter (OptiGene, UK); and (ii) real-time quantitative PCR (qPCR). The Loopamp test returned 98.8% (95% confidence interval-CI: 96.0-100.00) sensitivity and specificity of 97.7% (95% CI: 92.2-100) on VL samples, and 100% (95% CI: 99.1-100) sensitivity and 100.0% (95% CI: 98.8-100.0) specificity on CL samples. The Loopamp time-to-positivity (Tp) obtained by real-time fluorimetry showed excellent concordance (C = 97.91%) and strong correlation (r = 0.799) with qPCR's cycle threshold (Ct). The performance of Loopamp is comparable to that of LnPCR and qPCR in the diagnosis of cutaneous and visceral leishmaniasis due to L. infantum. The excellent correlation between the Tp and Ct should be further investigated to determine the accuracy of Loopamp to quantify parasite load in tissues.
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The increasing use of immunosuppressants in areas where visceral leishmaniasis (VL) is endemic has increased the number of people susceptible to developing more severe forms of the disease. Few studies have examined the quality of the immune response in immunosuppressed patients or experimental animals with VL. The present work characterises the parasite load developed in, and immune response to, Leishmania infantum-induced VL in C57BL/6 mice that, prior to and during infection, received immunosuppressant treatment with methylprednisolone (MPDN), anti-tumour necrosis factor (anti-TNF) antibodies, or methotrexate (MTX). The latter two treatments induced a significant reduction in the number of CD4+ T lymphocytes over the infection period. The anti-TNF treatment was also associated with a higher parasite load in the liver and a lower parasite load in the spleen. This, plus a possibly treatment-induced reduction in the number of cytokine-producing Th1 cells in the spleen, indicates the development of more severe VL. Interestingly, the MPDN and (especially) MTX treatments provoked a greater presence of soluble Leishmania antigen-specific multi-cytokine-producing T cells in the spleen and a lower liver parasite load than in control animals. These results highlight the need to better understand how immunosuppressant treatments might influence the severity of VL in human patients.
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Imunidade , Imunossupressores , Leishmaniose Visceral/imunologia , Carga Parasitária , Animais , Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos , Citocinas/imunologia , Feminino , Humanos , Imunidade Celular , Imunoglobulina G , Leishmania infantum/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia , Linfócitos T , Inibidores do Fator de Necrose Tumoral , Fator de Necrose Tumoral alfa/imunologiaRESUMO
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.
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Citocinas/metabolismo , Leishmania donovani/fisiologia , Leishmaniose Visceral/imunologia , Adolescente , Adulto , Células Cultivadas , Estudos de Coortes , Doenças Endêmicas , Etiópia/epidemiologia , Feminino , Seguimentos , Humanos , Imunização , Leishmaniose Visceral/epidemiologia , Masculino , Monitorização Fisiológica , Projetos Piloto , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: In 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 FINDINGS: Asymptomatic 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 / SIGNIFICANCE: There 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'.
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Reservatórios de Doenças , Transmissão de Doença Infecciosa , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/transmissão , Adulto , Animais , Anticorpos Antiprotozoários/sangue , Doenças Assintomáticas/epidemiologia , DNA de Protozoário/sangue , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Psychodidae/parasitologia , Espanha/epidemiologiaRESUMO
Progress has been made in the control or elimination of tropical diseases, with a significant reduction of incidence. However, there is a risk of re-emergence if the factors fueling transmission are not dealt with. Although it is essential to understand these underlying factors for each disease, asymptomatic carriers are a common element that may promote resurgence; their impact in terms of proportion in the population and role in transmission needs to be determined. In this paper, we review the current evidence on whether or not to treat asymptomatic carriers given the relevance of their role in the transmission of a specific disease, the efficacy and toxicity of existing drugs, the Public Health interest, and the benefit at an individual level, for example, in Chagas disease, to prevent irreversible organ damage. In the absence of other control tools such as vaccines, there is a need for safer drugs with good risk/benefit profiles in order to change the paradigm so that it addresses the complete infectious process beyond manifest disease to include treatment of non-symptomatic infected persons.