Publications by authors named "Nancy Gore Saravia"

38 Publications

Adaptation and performance of a mobile application for early detection of cutaneous leishmaniasis.

PLoS Negl Trop Dis 2021 Feb 11;15(2):e0008989. Epub 2021 Feb 11.

University of California, San Diego, California, United States of America.

Background: Detection and management of neglected tropical diseases such as cutaneous leishmaniasis present unmet challenges stemming from their prevalence in remote, rural, resource constrained areas having limited access to health services. These challenges are frequently compounded by armed conflict or illicit extractive industries. The use of mobile health technologies has shown promise in such settings, yet data on outcomes in the field remain scarce.

Methods: We adapted a validated prediction rule for the presumptive diagnosis of CL to create a mobile application for use by community health volunteers. We used human-centered design practices and agile development for app iteration. We tested the application in three rural areas where cutaneous leishmaniasis is endemic and an urban setting where patients seek medical attention in the municipality of Tumaco, Colombia. The application was assessed for usability, sensitivity and inter-rater reliability (kappa) when used by community health volunteers (CHV), health workers and a general practitioner, study physician.

Results: The application was readily used and understood. Among 122 screened cases with cutaneous ulcers, sensitivity to detect parasitologically proven CL was >95%. The proportion of participants with parasitologically confirmed CL was high (88%), precluding evaluation of specificity, and driving a high level of crude agreement between the app and parasitological diagnosis. The chance-adjusted agreement (kappa) varied across the components of the risk score. Time to diagnosis was reduced significantly, from 8 to 4 weeks on average when CHV conducted active case detection using the application, compared to passive case detection by health facility-based personnel.

Conclusions: Translating a validated prediction rule to a mHealth technology has shown the potential to improve the capacity of community health workers and healthcare personnel to provide opportune care, and access to health services for underserved populations. These findings support the use of mHealth tools for NTD research and healthcare.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0008989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904137PMC
February 2021

Exploring social innovation in health in Central America and the Caribbean.

Rev Panam Salud Publica 2020 6;44:e77. Epub 2020 Aug 6.

Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM) and Universidad Icesi Cali Colombia Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM) and Universidad Icesi, Cali, Colombia.

Universal health coverage is a public health priority in the Americas. Social innovation in health offers novel solutions to unmet needs, by enabling health care delivery to be more inclusive, affordable, and effective. In 2017, an international collaborative consortium launched an open call for solutions that sought to identify social innovations in health in Central America and the Caribbean. The focus was set on how these solutions can strengthen health care delivery, with emphasis on reducing the impact of neglected transmissible diseases. A crowdsourcing strategy was implemented to identify social innovations in health. These were evaluated by an external panel of experts and practitioners and civil society representing the health and social innovation sectors, based on the appropriateness, innovativeness, and affordability of the solution. The three top-scoring solutions were analyzed through case studies including site visits by a team of investigators. Two key findings emerged from the response to the call: 1) innovative solutions were based on the knowledge and experience of individuals and communities facing adverse situations; 2) this knowledge was shared through health promotion and education, leading to empowerment of the communities. The principal challenges addressed by the solutions were the limited access to quality health care services and failed traditional strategies for vector control. The solutions identified demonstrated how social innovation can strengthen health systems by delivering novel solutions to health needs and articulating communities to enable them to work hand-in-hand with the health system toward universal health.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.26633/RPSP.2020.77DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406126PMC
August 2020

Profiles of Local and Systemic Inflammation in the Outcome of Treatment of Human Cutaneous Leishmaniasis Caused by ().

Infect Immun 2020 02 20;88(3). Epub 2020 Feb 20.

Centro Internacional de Entrenamiento e Investigaciones Médicas-CIDEIM, Cali, Colombia

The immune mechanisms that contribute to the efficacy of treatment of cutaneous leishmaniasis (CL) are not fully understood. The aim of this study was to define immune correlates of the outcome of treatment of CL caused by () species during standard of care treatment with pentavalent antimonials. We conducted a comparative expression profiling of immune response genes in peripheral blood mononuclear cells (PBMCs) and lesion biopsy specimens obtained from CL patients before and at the end of treatment (EoT) with meglumine antimoniate. The response of PBMCs to () partially reflected that of lesion microenvironments. Significant downregulation of gene expression profiles consistent with local innate immune responses (monocyte and neutrophil activation and chemoattractant molecules) was observed at EoT in biopsy specimens of patients who cured ( = 8), compared to those from patients with treatment failure ( = 8). Among differentially expressed genes, pretreatment expression of was significantly predictive of the therapeutic response (receiver operating characteristic [ROC] curve, area under the curve [AUC] = 0.82, = 0.02). Polymorphisms in regulatory regions of the promoter were analyzed in a pilot cohort of DNA samples from CL patients (cures,  = 20, and treatment failure,  = 20), showing putative association of polymorphisms rs13900(C/T) and rs2857656(G/C) with treatment outcome. Our data indicate that dampening gene expression profiles of monocyte and neutrophil activation characterize clinical cure after treatment of CL, supporting participation of parasite-sustained inflammation or deregulated innate immune responses in treatment failure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/IAI.00764-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035935PMC
February 2020

Building research capacity through "Planning for Success".

PLoS Negl Trop Dis 2019 08 1;13(8):e0007426. Epub 2019 Aug 1.

Centro Internacional de Entrenamiento e Investigaciones Médicas, Cali, Colombia.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0007426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675041PMC
August 2019

Resistance of to Meglumine Antimoniate or Miltefosine Modulates Neutrophil Effector Functions.

Front Immunol 2018 21;9:3040. Epub 2018 Dec 21.

Department of Biochemistry, WHO-Immunology Research and Training Center, University of Lausanne, Epalinges, Switzerland.

is the main causative agent of cutaneous leishmaniasis in Colombia and is usually treated with either meglumine antimoniate (MA) or miltefosine (MIL). In recent years, there has been increasing evidence of the emergence of drug-resistance against these compounds. Neutrophils are known to play an important role in immunity against . These cells are rapidly recruited upon infection and are also present in chronic lesions. However, their involvement in the outcome of infection with drug-resistant has not been examined. In this study, human and murine neutrophils were infected with MA or MIL drug-resistant . lines derived from a parental . drug-susceptible strain. Neutrophil effector functions were assessed analyzing the production of reactive oxygen species (ROS), the formation of neutrophil extracellular trap (NET) and the expression of cell surface activation markers. Parasite killing by neutrophils was assessed using . transfected with a luciferase reporter. We show here that MA and MIL-resistant . lines elicited significantly increased NET formation and MA-resistant . induced significantly increased ROS production in both murine and human neutrophils, compared to infections with the parental MIL and MA susceptible strain. Furthermore, neutrophils exposed to drug-resistant lines showed increased activation, as revealed by decreased expression of CD62L and increased expression of CD66b in human neutrophils yet presented higher survival within neutrophils than the drug-susceptible strain. These results provide evidence that parasite drug-susceptibility may influences neutrophil activation and function as well as parasite survival within neutrophils. Further investigaton of the inter-relationship of drug susceptibility and neutrophil effector function should contribute to better understanding of the factors involved in susceptibility to anti- drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2018.03040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6308327PMC
October 2019

Developing mobile health applications for neglected tropical disease research.

PLoS Negl Trop Dis 2018 11 1;12(11):e0006791. Epub 2018 Nov 1.

University of California, San Diego, California, United States of America.

Mobile applications (apps) can bring health research and its potential downstream benefits closer to underserved populations. Drawing on experience developing an app for detecting and referring cases of cutaneous leishmaniasis in Colombia, called Guaral/app, we review key steps in creating such mobile health (mHealth) tools. These require consideration of the sociotechnical context using methods such as systems analysis and human-centered design (HCD), predicated on engagement and iteration with all stakeholders. We emphasize usability and technical concerns and describe the interdependency of technical and human considerations for mHealth systems in rural communities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0006791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211619PMC
November 2018

Strengthening institutional capacity for equitable health research: lessons from Latin America and the Caribbean.

BMJ 2018 Jul 16;362:k2456. Epub 2018 Jul 16.

Health Services and Access Unit, Department of Health Systems and Services of the Pan American Health Organization/World Health Organization, Washington DC, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6046649PMC
http://dx.doi.org/10.1136/bmj.k2456DOI Listing
July 2018

Simultaneous population pharmacokinetic modelling of plasma and intracellular PBMC miltefosine concentrations in New World cutaneous leishmaniasis and exploration of exposure-response relationships.

J Antimicrob Chemother 2018 08;73(8):2104-2111

Department of Pharmacy & Pharmacology, Antoni van Leeuwenhoek Hospital/The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Objectives: Leishmania parasites reside within macrophages and the direct target of antileishmanial drugs is therefore intracellular. We aimed to characterize the intracellular PBMC miltefosine kinetics by developing a population pharmacokinetic (PK) model simultaneously describing plasma and intracellular PBMC pharmacokinetics. Furthermore, we explored exposure-response relationships and simulated alternative dosing regimens.

Patients And Methods: A population PK model was developed with NONMEM, based on 339 plasma and 194 PBMC miltefosine concentrations from Colombian cutaneous leishmaniasis patients [29 children (2-12 years old) and 22 adults] receiving 1.8-2.5 mg/kg/day miltefosine for 28 days.

Results: A three-compartment model with miltefosine distribution into an intracellular PBMC effect compartment best fitted the data. Intracellular PBMC distribution was described with an intracellular-to-plasma concentration ratio of 2.17 [relative standard error (RSE) 4.9%] and intracellular distribution rate constant of 1.23 day-1 (RSE 14%). In exploring exposure-response relationships, both plasma and intracellular model-based exposure estimates significantly influenced probability of cure. A proposed PK target for the area under the plasma concentration-time curve (day 0-28) of >535 mg·day/L corresponded to >95% probability of cure. In linear dosing simulations, 18.3% of children compared with 2.8% of adults failed to reach 535 mg·day/L. In children, this decreased to 1.8% after allometric dosing simulation.

Conclusions: The developed population PK model described the rate and extent of miltefosine distribution from plasma into PBMCs. Miltefosine exposure was significantly related to probability of cure in this cutaneous leishmaniasis patient population. We propose an exploratory PK target, which should be validated in a larger cohort study.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jac/dky143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251527PMC
August 2018

Harmonized clinical trial methodologies for localized cutaneous leishmaniasis and potential for extensive network with capacities for clinical evaluation.

PLoS Negl Trop Dis 2018 01 12;12(1):e0006141. Epub 2018 Jan 12.

Drugs for Neglected Diseases initiative (DNDi), Geneva, Switzerland.

Introduction: Progress with the treatment of cutaneous leishmaniasis (CL) has been hampered by inconsistent methodologies used to assess treatment effects. A sizable number of trials conducted over the years has generated only weak evidence backing current treatment recommendations, as shown by systematic reviews on old-world and new-world CL (OWCL and NWCL).

Materials And Methods: Using a previously published guidance paper on CL treatment trial methodology as the reference, consensus was sought on key parameters including core eligibility and outcome measures, among OWCL (7 countries, 10 trial sites) and NWCL (7 countries, 11 trial sites) during two separate meetings.

Results: Findings and level of consensus within and between OWCL and NWCL sites are presented and discussed. In addition, CL trial site characteristics and capacities are summarized.

Conclusions: The consensus reached allows standardization of future clinical research across OWCL and NWCL sites. We encourage CL researchers to adopt and adapt as required the proposed parameters and outcomes in their future trials and provide feedback on their experience. The expertise afforded between the two sets of clinical sites provides the basis for a powerful consortium with potential for extensive, standardized assessment of interventions for CL and faster approval of candidate treatments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0006141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785032PMC
January 2018

Pathophysiology of Leishmania Infection during Pregnancy.

Trends Parasitol 2017 12 4;33(12):935-946. Epub 2017 Oct 4.

Instituto Nacional de Perinatología Isidro Espinosa de los Reyes, Mexico City, Mexico.

The pathological processes resulting from parasitic infection are known to have important impacts on the mother child dyad during pregnancy. The roles of parasite transmission and the maternal immune response have been described in diseases such as malaria, toxoplasmosis, and trypanosomiasis. However, the impact of parasites of the genus Leishmania, etiological agents of the neglected tropical diseases tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL), is comparatively less well known, though it is an increasingly recognized concern for infected mothers and their fetuses. In this review, we first consider the pathophysiology of placental infection and transplacental transmission of this parasite, and then discuss the role and mechanisms of the maternal immune system in simultaneously mediating maternal-fetal infection and adverse pregnancy outcomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pt.2017.08.012DOI Listing
December 2017

Clinical and parasitological factors in parasite persistence after treatment and clinical cure of cutaneous leishmaniasis.

PLoS Negl Trop Dis 2017 Jul 13;11(7):e0005713. Epub 2017 Jul 13.

Centro Internacional de Entrenamiento e Investigaciones Médicas-CIDEIM, Cali, Colombia.

Background: The determinants of parasite persistence or elimination after treatment and clinical resolution of cutaneous leishmaniasis (CL) are unknown. We investigated clinical and parasitological parameters associated with the presence and viability of Leishmania after treatment and resolution of CL caused by L. Viannia.

Methods: Seventy patients who were treated with meglumine antimoniate (n = 38) or miltefosine (n = 32) and cured, were included in this study. Leishmania persistence and viability were determined by detection of kDNA and 7SLRNA transcripts, respectively, before, at the end of treatment (EoT), and 13 weeks after initiation of treatment in lesions and swabs of nasal and tonsillar mucosa.

Results: Sixty percent of patients (42/70) had evidence of Leishmania persistence at EoT and 30% (9/30) 13 weeks after treatment initiation. A previous episode of CL was found to be a protective factor for detectable Leishmania persistence (OR: 0.16, 95%CI: 0.03-0.92). kDNA genotyping could not discern differences between parasite populations that persisted and those isolated at diagnosis.

Conclusions: Leishmania persist in skin and mucosal tissues in a high proportion of patients who achieved therapeutic cure of CL. This finding prompts assessment of the contribution of persistent infection in transmission and endemicity of CL, and in disease reactivation and protective immunity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0005713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526576PMC
July 2017

Cost-effectiveness of meglumine antimoniate versus miltefosine caregiver DOT for the treatment of pediatric cutaneous leishmaniasis.

PLoS Negl Trop Dis 2017 04 6;11(4):e0005459. Epub 2017 Apr 6.

Department of Medicine, Section of Infectious Diseases and Global Health, University of Chicago, Chicago, Illinois, United States of America.

Background: Oral miltefosine has been shown to be non-inferior to first-line, injectable meglumine antimoniate (MA) for the treatment of cutaneous leishmaniasis (CL) in children. Miltefosine may be administered via in-home caregiver Directly Observed Therapy (cDOT), while patients must travel to clinics to receive MA. We performed a cost-effectiveness analysis comparing miltefosine by cDOT versus MA for pediatric CL in southwest Colombia.

Methodology/principle Findings: We developed a Monte Carlo model comparing the cost-per-cure of miltefosine by cDOT compared to MA from patient, government payer, and societal perspectives (societal = sum of patient and government payer perspective costs). Drug effectiveness and adverse events were estimated from clinical trials. Healthcare utilization and costs of travel were obtained from surveys of providers and published sources. The primary outcome was cost-per-cure reported in 2015 USD. Treatment efficacy, costs, and adherence were varied in sensitivity analysis to assess robustness of results. Treatment with miltefosine resulted in substantially lower cost-per-cure from a societal and patient perspective, and slightly higher cost-per-cure from a government payer perspective compared to MA. Mean societal cost-per-cure were $531 (SD±$239) for MA and $188 (SD±$100) for miltefosine, a mean cost-per-cure difference of +$343. Mean cost-per-cure from a patient perspective were $442 (SD ±$233) for MA and $30 (SD±$16) for miltefosine, a mean difference of +$412. Mean cost-per-cure from a government perspective were $89 (SD±$55) for MA and $158 (SD±$98) for miltefosine, with a mean difference of -$69. Results were robust across a variety of assumptions in univariate and multi-way analysis.

Conclusions/significance: Treatment of pediatric cutaneous leishmaniasis with miltefosine via cDOT is cost saving from patient and societal perspectives, and moderately more costly from the government payer perspective compared to treatment with MA. Results were robust over a range of sensitivity analyses. Lower drug price for miltefosine could result in cost saving from a government perspective.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0005459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5404883PMC
April 2017

Local Delivery of the Toll-Like Receptor 9 Ligand CpG Downregulates Host Immune and Inflammatory Responses, Ameliorating Established Leishmania (Viannia) panamensis Chronic Infection.

Infect Immun 2017 03 23;85(3). Epub 2017 Feb 23.

Yale University School of Public Health, New Haven, Connecticut, USA.

Infection by () , the predominant etiologic agent for cutaneous leishmaniasis in Colombia, is characterized by a chronic mixed inflammatory response. Current treatment options are plagued by toxicity, lengthy treatment regimens, and growing evidence of drug resistance. Immunotherapy, modulating the immune system to mount a protective response, may provide an alternate therapeutic approach. We investigated the ability of the Toll-like receptor 9 (TLR9) ligand CpG to modulate established disease in the () mouse model. Treatment of established infection with a high dose (50 μg) of CpG ameliorated disease and lowered parasite burden. Interestingly, immediately after treatment there was a significant increase in transforming growth factor β (TGF-β) and concomitantly an increase in T regulatory cell (Treg) function. Although a general reduction in cell-mediated immune cytokine and chemokine (gamma interferon [IFN-γ], interleukin 10 [IL-10], IL-13, IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-4, and MIP-1α) responses of the treated mice was observed, certain chemokines (RANTES, monocyte chemoattractant protein 1[MCP-1], and IP-10) were increased. Further, in peripheral blood mononuclear cells (PBMCs) from patients with cutaneous leishmaniasis, CpG treatment similarly exhibited a dose-response effect on the production of IFN-γ, IL-17, IL-10, and IL-13, with reductions observed at higher doses. To further understand the underlying mechanisms and cell populations driving the CpG mediated response, we examined the dose effects mediated by the TLR9 cell populations (dendritic cells, macrophages, and B cells) found to accumulate labeled CpG Notably, B cells altered the production of IL-17, IL-13, and IFN-γ, supporting a role for B cells functioning as antigen-presenting cells (APCs) and/or regulatory cells during infection. Interestingly, B cells have been previously demonstrated as a primary type of APC in patients infected with () and thus may be useful targets of immunotherapy. Collectively, our results show that CpG-induced immune regulation leads to a dampening of the host immune response and healing in the mouse model, and it may provide an alternate approach to treatment of cutaneous leishmaniasis caused by () .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/IAI.00981-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5328479PMC
March 2017

Pharmacokinetics of Miltefosine in Children and Adults with Cutaneous Leishmaniasis.

Antimicrob Agents Chemother 2017 03 23;61(3). Epub 2017 Feb 23.

Centro Internacional de Entrenamiento e Investigaciones Médicas, Cali, Colombia

An open-label pharmacokinetics (PK) clinical trial was conducted to comparatively assess the PK and explore the pharmacodynamics (PD) of miltefosine in children and adults with cutaneous leishmaniasis (CL) in Colombia. Sixty patients, 30 children aged 2 to 12 years and 30 adults aged 18 to 60 years, were enrolled. Participants received miltefosine (Impavido) at a nominal dose of 2.5 mg/kg/day for 28 days. Miltefosine concentrations were measured in plasma and peripheral blood mononuclear cells by liquid chromatography-tandem mass spectrometry of samples obtained during treatment and up to 6 months following completion of treatment, when therapeutic outcome was determined. Fifty-two patients were cured, 5 pediatric patients failed treatment, and 3 participants were lost to follow-up. () predominated among the strains isolated (42/46; 91%). Noncompartmental analysis demonstrated that plasma and intracellular miltefosine concentrations were, overall, lower in children than in adults. Exposure to miltefosine, estimated by area under the concentration-time curve and maximum concentration, was significantly lower in children in both the central and intracellular compartments ( < 0.01). persistence was detected in 43% of study participants at the end of treatment and in 27% at 90 days after initiation of treatment. Clinical response was not dependent on parasite elimination. miltefosine susceptibility was similar for strains from adults and children. Our results document PK differences for miltefosine in children and adults with cutaneous leishmaniasis that affect drug exposure and could influence the outcome of treatment, and they provide bases for optimizing therapeutic regimens for CL in pediatric populations. (This study has been registered at ClinicalTrials.gov under identifier NCT01462500.).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.02198-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5328512PMC
March 2017

Ex vivo host and parasite response to antileishmanial drugs and immunomodulators.

PLoS Negl Trop Dis 2015 May 29;9(5):e0003820. Epub 2015 May 29.

Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), Cali, Colombia.

Background: Therapeutic response in infectious disease involves host as well as microbial determinants. Because the immune and inflammatory response to Leishmania (Viannia) species defines the outcome of infection and efficacy of treatment, immunomodulation is considered a promising therapeutic strategy. However, since Leishmania infection and antileishmanial drugs can themselves modulate drug transport, metabolism and/or immune responses, immunotherapeutic approaches require integrated assessment of host and parasite responses.

Methodology: To achieve an integrated assessment of current and innovative therapeutic strategies, we determined host and parasite responses to miltefosine and meglumine antimoniate alone and in combination with pentoxifylline or CpG 2006 in peripheral blood mononuclear cells (PBMCs) of cutaneous leishmaniasis patients. Parasite survival and secretion of TNF-α, IFN-γ, IL-10 and IL-13 were evaluated concomitantly in PBMCs infected with Luc-L. (V.) panamensis exposed to meglumine antimoniate (4, 8, 16, 32 and 64 μg SbV/mL) or miltefosine (2, 4, 8, 16 and 32 μM HePC). Concentrations of 4 μM of miltefosine and 8 μg SbV/mL were selected for evaluation in combination with immunomodulators based on the high but partial reduction of parasite burden by these antileishmanial concentrations without affecting cytokine secretion of infected PBMCs. Intracellular parasite survival was determined by luminometry and cytokine secretion measured by ELISA and multiplex assays.

Principal Findings: Anti- and pro-inflammatory cytokines characteristic of L. (V.) panamensis infection were evaluable concomitantly with viability of Leishmania within monocyte-derived macrophages present in PBMC cultures. Both antileishmanial drugs reduced the parasite load of macrophages; miltefosine also suppressed IL-10 and IL-13 secretion in a dose dependent manner. Pentoxifylline did not affect parasite survival or alter antileishmanial effects of miltefosine or meglumine antimoniate. However, pentoxifylline diminished secretion of TNF-α, IFN-γ and IL-13, cytokines associated with the outcome of infection by species of the Viannia subgenus. Exposure to CpG diminished the leishmanicidal effect of meglumine antimoniate, but not miltefosine, and significantly reduced secretion of IL-10, alone and in combination with either antileishmanial drug. IL-13 increased in response to CpG plus miltefosine.

Conclusions And Significance: Human PBMCs allow integrated ex vivo assessment of antileishmanial treatments, providing information on host and parasite determinants of therapeutic response that may be used to tailor therapeutic strategies to optimize clinical resolution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0003820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4449175PMC
May 2015

First report of Warileya rotundipennis (Psychodidae: Phlebotominae) naturally infected with Leishmania (Viannia) in a focus of cutaneous leishmaniasis in Colombia.

Acta Trop 2015 Aug 25;148:191-6. Epub 2015 Apr 25.

Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), Carrera 125 No. 19-225 Avenida La María-Pance, Cali Código Postal 760031, Colombia. Electronic address:

The expansion of transmission of cutaneous leishmaniasis from sylvatic ecosystems into peri-urban and domestic settings has occurred as sand flies have adapted to anthropogenic environmental modifications. Assessment of the intradomiciliary presence of sand flies in households of the settlement "La Cabaña", in the Department of Risaralda, Colombia, revealed an abundance of Warileya rotundipennis. This unexpected observation motivated further analyses to evaluate the participation of this species in the transmission of cutaneous leishmaniasis. Collections using CDC light traps were conducted during two consecutive nights in May and August 2011.The total of 667 sand flies collected were classified into five species: W. rotundipennis (n=654; 98.05%), Nyssomyia trapidoi (n=7; 1.04%); Lutzomyia (Helcocyrtomyia) hartmanni (n=3; 0.44%); Lutzomyia lichyi (n=2; 0.29%) and Psychodopygus panamensis (n=1; 0.14%). The striking predominance of W. rotundipennis within households during both wet (May) and dry (August) seasons, anthropophilic behavior demonstrated by human blood in 95.23% (60/63) evaluable blood-engorged specimens, and natural infection (5/168-3%) with genetically similar parasites of the Leishmania (Viannia) subgenus observed in a patient in this community, support the involvement of W. rotundipennis in the domestic transmission of cutaneous leishmaniasis in "La Cabaña".
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.actatropica.2015.04.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4654406PMC
August 2015

Sensitive diagnosis of cutaneous leishmaniasis by lesion swab sampling coupled to qPCR.

Parasitology 2014 Dec 11;141(14):1891-7. Epub 2014 Aug 11.

Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM),Cra 125 # 19-225, Cali,Colombia.

Variation in clinical accuracy of molecular diagnostic methods for cutaneous leishmaniasis (CL) is commonly observed depending on the sample source, the method of DNA recovery and the molecular test. Few attempts have been made to compare these variables. Two swab and aspirate samples from lesions of patients with suspected CL (n = 105) were evaluated alongside standard diagnosis by microscopic detection of amastigotes or culture of parasites from lesion material. Three DNA extraction methods were compared: Qiagen on swab and aspirate specimens, Isohelix on swabs and Boil/Spin of lesion aspirates. Recovery of Leishmania DNA was evaluated for each sample type by real-time polymerase chain reaction detection of parasitic 18S rDNA, and the diagnostic accuracy of the molecular method determined. Swab sampling combined with Qiagen DNA extraction was the most efficient recovery method for Leishmania DNA, and was the most sensitive (98%; 95% CI: 91-100%) and specific (84%; 95% CI: 64-95%) approach. Aspirated material was less sensitive at 80% (95% CI: 70-88%) and 61% (95% CI: 50-72%) when coupled to Qiagen or Boil-Spin DNA extraction, respectively. Swab sampling of lesions was painless, simple to perform and coupled with standardized DNA extraction enhances the feasibility of molecular diagnosis of CL.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1017/S0031182014001280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4654403PMC
December 2014

Miltefosine and antimonial drug susceptibility of Leishmania Viannia species and populations in regions of high transmission in Colombia.

PLoS Negl Trop Dis 2014 May 22;8(5):e2871. Epub 2014 May 22.

Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), Cali, Colombia.

Background: Pentavalent antimonials have been the first line treatment for dermal leishmaniasis in Colombia for over 30 years. Miltefosine is administered as second line treatment since 2005. The susceptibility of circulating populations of Leishmania to these drugs is unknown despite clinical evidence supporting the emergence of resistance.

Methodology/principal Findings: In vitro susceptibility was determined for intracellular amastigotes of 245 clinical strains of the most prevalent Leishmania Viannia species in Colombia to miltefosine (HePC) and/or meglumine antimoniate (Sb(V)); 163, (80%) were evaluated for both drugs. Additionally, susceptibility to Sb(V) was examined in two cohorts of 85 L. V. panamensis strains isolated between 1980-1989 and 2000-2009 in the municipality of Tumaco. Susceptibility to each drug differed among strains of the same species and between species. Whereas 68% of L. V. braziliensis strains presented in vitro resistance to HePC, 69% were sensitive to Sb(V). Resistance to HePC and Sb(V) occurred respectively, in 20% y 21% of L. panamensis strains. Only 3% of L. V. guyanensis were resistant to HePC, and none to Sb(V). Drug susceptibility differed between geographic regions and time periods. Subpopulations having disparate susceptibility to Sb(V) were discerned among L. V. panamensis strains isolated during 1980-1990 in Tumaco where resistant strains belonged to zymodeme 2.3, and sensitive strains to zymodeme 2.2.

Conclusions/significance: Large scale evaluation of clinical strains of Leishmania Viannia species demonstrated species, population, geographic, and epidemiologic differences in susceptibility to meglumine antimoniate and miltefosine, and provided baseline information for monitoring susceptibility to these drugs. Sensitive and resistant clinical strains within each species, and zymodeme as a proxy marker of antimony susceptibility for L. V. panamensis, will be useful in deciphering factors involved in susceptibility and the distribution of sensitive and resistant populations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0002871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4031164PMC
May 2014

Chronicity of dermal leishmaniasis caused by Leishmania panamensis is associated with parasite-mediated induction of chemokine gene expression.

Infect Immun 2014 Jul 21;82(7):2872-80. Epub 2014 Apr 21.

Centro Internacional de Entrenamiento e Investigaciones Médicas, Cali, Colombia

Chronic tegumentary leishmaniasis is characterized by a scarcity of parasites in lesions and a heightened inflammatory response. Deregulated and hyperactive inflammation contributes to tissue damage and parasite persistence. The mechanisms by which immune cells are recruited to the lesion and their relationship to clinical outcomes remain elusive. We examined the expression levels of chemokines and their receptors in relation to clinical outcome in dermal leishmaniasis caused by Leishmania (Viannia) panamensis. Primary macrophages from healthy donors were infected with L. panamensis strains isolated from self-healing patients (n = 4) and those presenting chronic disease (n = 5). A consistent pattern of upregulation of neutrophil (cxcl1, cxcl2, cxcl5, and cxcl8/il-8) and monocyte (ccl2, ccl7, ccl8, cxcl3, and cxcl10) chemotactic chemokines and ccr1 and ccr5 receptor genes, evaluated by reverse transcription-quantitative PCR (qRT-PCR), was observed upon infection with strains from patients with chronic dermal leishmaniasis; induction of CXCL5 and CCL8 was corroborated at the protein level. No apparent upregulation was elicited in macrophages infected with strains from self-healing patients. Expression levels of ccl8, cxcl2, cxcl3, and cxcl5 in lesion biopsy specimens from patients with chronic cutaneous leishmaniasis (CL) were compared to those in biopsy specimens from Montenegro skin tests of individuals with asymptomatic infection. Increased expression levels of cxcl5 (P < 0.05), ccl8, and cxcl3 were corroborated in chronic CL lesions. Our study revealed a dichotomy in macrophage chemokine gene expression elicited by L. panamensis strains from patients with self-healing disease and those presenting chronic disease, consistent with parasite-mediated hyperactivation of the inflammatory response driving chronicity. The predominant upregulation of neutrophil and monocyte chemoattractants indicates novel mechanisms of sustained inflammatory activation and may provide new therapeutic targets against chronic dermal leishmaniasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/IAI.01133-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097649PMC
July 2014

CD4 T cell activation by B cells in human Leishmania (Viannia) infection.

BMC Infect Dis 2014 Feb 25;14:108. Epub 2014 Feb 25.

Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), Cali, Colombia.

Background: An effective adaptive immune response requires activation of specific CD4 T cells. The capacity of B cells to activate CD4 T cells in human cutaneous leishmaniasis caused by Leishmania (Viannia) has not been evaluated.

Methods: CD4 T cell activation by B cells of cutaneous leishmaniasis patients was evaluated by culture of PBMCs or purified B cells and CD4 T cells with Leishmania panamensis antigens. CD4 T cell and B cell activation markers were evaluated by flow cytometry and 13 cytokines were measured in supernatants with a bead-based capture assay. The effect of Leishmania antigens on BCR-mediated endocytosis of ovalbumin was evaluated in the Ramos human B cell line by targeting the antigen with anti-IgM-biotin and anti-biotin-ovalbumin-FITC.

Results: Culture of PBMCs from cutaneous leishmaniasis patients with Leishmania antigens resulted in upregulation of the activation markers CD25 and CD69 as well as increased frequency of CD25hiCD127- cells among CD4 T cells. Concomitantly, B cells upregulated the costimulatory molecule CD86. These changes were not observed in PBMCs from healthy subjects, indicating participation of Leishmania-specific lymphocytes expanded in vivo. Purified B cells from these patients, when interacting with purified CD4 T cells and Leishmania antigens, were capable of inducing significant increases in CD25 and CD69 expression and CD25hiCD127- frequency in CD4 T cells. These changes were associated with upregulation of CD86 in B cells. Comparison of changes in CD4 T cell activation parameters between PBMC and B cell/CD4 T cell cultures showed no statistically significant differences; further, significant secretion of IFN-γ, TNF-α, IL-6 and IL-13 was induced in both types of cultures. Additionally, culture with Leishmania antigens enhanced BCR-mediated endocytosis of ovalbumin in Ramos human B cells.

Conclusions: The capacity of B cells specific for Leishmania antigens in peripheral blood of cutaneous leishmaniasis patients to activate CD4 T cells and induce cytokine secretion is similar to that of all cell populations present in PBMCs. This capacity implicates B cells as a plausible target for modulation of the immune response to Leishmania infection as a therapeutic strategy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2334-14-108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937821PMC
February 2014

Treatment failure and miltefosine susceptibility in dermal leishmaniasis caused by Leishmania subgenus Viannia species.

Antimicrob Agents Chemother 2014 21;58(1):144-52. Epub 2013 Oct 21.

Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), Cali, Colombia.

Treatment failure and parasite drug susceptibility in dermal leishmaniasis caused by Leishmania (Viannia) species are poorly understood. Prospective evaluation of drug susceptibility of strains isolated from individual patients before drug exposure and at clinical failure allows intrinsic and acquired differences in susceptibility to be discerned and analyzed. To determine whether intrinsic susceptibility or loss of susceptibility to miltefosine contributed to treatment failure, we evaluated the miltefosine susceptibility of intracellular amastigotes and promastigotes of six Leishmania (Viannia) braziliensis and six Leishmania (Viannia) panamensis strains isolated sequentially, at diagnosis and treatment failure, from two children and four adults ≥55 years old with concurrent conditions. Four patients presented only cutaneous lesions, one had mucosal disease, and one had disseminated mucocutaneous disease. Expression of the Leishmania drug transporter genes abca2, abca3, abcc2, abcc3, abcg4, abcg6, and LbMT was evaluated by quantitative reverse transcription-PCR (qRT-PCR). Intracellular amastigotes (median 50% effective concentration [EC50], 10.7 μmol/liter) were more susceptible to miltefosine than promastigotes (median EC50, 55.3 μmol/liter) (P < 0.0001). Loss of susceptibility at failure, demonstrated by a miltefosine EC50 of >32 μmol/liter (the upper limit of intracellular amastigote assay), occurred in L. panamensis infection in a child and in L. braziliensis infection in an adult and was accompanied by decreased expression of the miltefosine transporter LbMT (LbMT/β-tubulin, 0.42- to 0.26-fold [P = 0.039] and 0.70- to 0.57-fold [P = 0.009], respectively). LbMT gene polymorphisms were not associated with susceptibility phenotype. Leishmania ABCA3 transporter expression was inversely correlated with miltefosine susceptibility (r = -0.605; P = 0.037). Loss of susceptibility is one of multiple factors involved in failure of miltefosine treatment in dermal leishmaniasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.01023-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3910710PMC
September 2014

Leishmania panamensis infection and antimonial drugs modulate expression of macrophage drug transporters and metabolizing enzymes: impact on intracellular parasite survival.

J Antimicrob Chemother 2014 Jan 24;69(1):139-49. Epub 2013 Aug 24.

Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), Carrera 125 No. 19-225 Cali, Colombia.

Objectives: Treatment failure is multifactorial. Despite the importance of host cell drug transporters and metabolizing enzymes in the accumulation, distribution and metabolism of drugs targeting intracellular pathogens, their impact on the efficacy of antileishmanials is unknown. We examined the contribution of pharmacologically relevant determinants in human macrophages in the antimony-mediated killing of intracellular Leishmania panamensis and its relationship with the outcome of treatment with meglumine antimoniate.

Methods: Patients with cutaneous leishmaniasis who failed (n = 8) or responded (n =8) to treatment were recruited. Gene expression profiling of pharmacological determinants in primary macrophages was evaluated by quantitative RT-PCR and correlated to the drug-mediated intracellular parasite killing. Functional validation was conducted through short hairpin RNA gene knockdown.

Results: Survival of L. panamensis after exposure to antimonials was significantly higher in macrophages from patients who failed treatment. Sixteen macrophage drug-response genes were modulated by infection and exposure to meglumine antimoniate. Correlation analyses of gene expression and intracellular parasite survival revealed the involvement of host cell metallothionein-2A and ABCB6 in the survival of Leishmania during exposure to antimonials. ABCB6 was functionally validated as a transporter of antimonial compounds localized in both the cell and phagolysosomal membranes of macrophages, revealing a novel mechanism of host cell-mediated regulation of intracellular drug exposure and parasite survival within phagocytes.

Conclusions: These results provide insight into host cell mechanisms regulating the intracellular exposure of Leishmania to antimonials and variations among individuals that impact parasite survival. Understanding of host cell determinants of intracellular pharmacokinetics/pharmacodynamics opens new avenues to improved drug efficacy for intracellular pathogens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jac/dkt334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861331PMC
January 2014

Clinical and epidemiologic profile of cutaneous leishmaniasis in Colombian children: considerations for local treatment.

Am J Trop Med Hyg 2013 Aug 24;89(2):359-64. Epub 2013 Jun 24.

Centro Internacional de Entrenamiento e Investigaciones Medicas-CIDEIM (International Center for Medical Research and Training), Cali, Colombia.

Treatment alternatives have seldom been evaluated in children with cutaneous leishmaniasis (CL). We examine the clinical/epidemiological profile of children with CL considering international guidelines for local treatment. Descriptive analyses were conducted using International Center for Medical Research and Training (CIDEIM) case reports of parasitologically diagnosed patients ≤ 14 years of age from 2004 to 2010. Eligibility for local treatment based on World Health Organization/Pan American Health Organization (WHO/PAHO) criteria was determined. Among 380 children, 90% presented lesions of < 3 months duration, 54% presented single lesions < 30 mm in diameter, and 45% were ≤ 5 years old. Lesions on the head and neck were more frequent among children 0-5 years, and lesions below the head/neck were more frequent among 11- to 14-year-old children (P = 0.004). Using PAHO and WHO criteria, 26% and 53% of children, respectively, were eligible for local treatment. Recommended local treatments for New World CL have potential but limited applicability in children. Individual risk-benefit assessment and effectiveness data in children may increase eligibility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4269/ajtmh.12-0784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3741260PMC
August 2013

Detection of Leishmania RNA virus in Leishmania parasites.

PLoS Negl Trop Dis 2013 10;7(1):e2006. Epub 2013 Jan 10.

Department of Biochemistry, University of Lausanne, Epalinges, Vaud, Switzerland.

Background: Patients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence.

Methodology/principal Findings: This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice.

Conclusions/significance: We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0002006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542153PMC
June 2013

Regulatory T cells in the pathogenesis and healing of chronic human dermal leishmaniasis caused by Leishmania (Viannia) species.

PLoS Negl Trop Dis 2012 24;6(4):e1627. Epub 2012 Apr 24.

Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), Cali, Colombia.

Background: The inflammatory response is prominent in the pathogenesis of dermal leishmaniasis. We hypothesized that regulatory T cells (Tregs) may be diminished in chronic dermal leishmaniasis (CDL) and contribute to healing during treatment.

Methodology/principal Findings: The frequency and functional capacity of Tregs were evaluated at diagnosis and following treatment of CDL patients having lesions of ≥6 months duration and asymptomatically infected residents of endemic foci. The frequency of CD4(+)CD25(hi) cells expressing Foxp3 or GITR or lacking expression of CD127 in peripheral blood was determined by flow cytometry. The capacity of CD4(+)CD25(+) cells to inhibit Leishmania-specific responses was determined by co-culture with effector CD4(+)CD25(-) cells. The expression of FOXP3, IFNG, IL10 and IDO was determined in lesion and leishmanin skin test site biopsies by qRT-PCR. Although CDL patients presented higher frequency of CD4(+)CD25(hi)Foxp3(+) cells in peripheral blood and higher expression of FOXP3 at leishmanin skin test sites, their CD4(+)CD25(+) cells were significantly less capable of suppressing antigen specific-IFN-γ secretion by effector cells compared with asymptomatically infected individuals. At the end of treatment, both the frequency of CD4(+)CD25(hi)CD127(-) cells and their capacity to inhibit proliferation and IFN-γ secretion increased and coincided with healing of cutaneous lesions. IDO was downregulated during healing of lesions and its expression was positively correlated with IFNG but not FOXP3.

Conclusions/significance: The disparity between CD25(hi)Foxp3(+) CD4 T cell frequency in peripheral blood, Foxp3 expression at the site of cutaneous responses to leishmanin, and suppressive capacity provides evidence of impaired Treg function in the pathogenesis of CDL. Moreover, the concurrence of increased Leishmania-specific suppressive capacity with induction of a CD25(hi)CD127(-) subset of CD4 T cells during healing supports the participation of Tregs in the resolution of chronic dermal lesions. Treg subsets may therefore be relevant in designing immunotherapeutic strategies for recalcitrant dermal leishmaniasis caused by Leishmania (Viannia) species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0001627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3335885PMC
August 2012

Novel approach to in vitro drug susceptibility assessment of clinical strains of Leishmania spp.

J Clin Microbiol 2012 Jul 18;50(7):2207-11. Epub 2012 Apr 18.

Centro Internacional de Entrenamiento e Investigaciones Médica, Cali, Colombia.

Resistance to antimonial drugs has been documented in Leishmania isolates transmitted in South America, Europe, and Asia. The frequency and distribution of resistance to these and other antileishmanial drugs are unknown. Technical constraints have limited the assessment of drug susceptibility of clinical strains of Leishmania. Susceptibility of experimentally selected lines and 130 clinical strains of Leishmania panamensis, L. braziliensis, and L. guyanensis to meglumine antimoniate and miltefosine was determined on the basis of parasite burden and percentage of infected U-937 human macrophages. Reductions of infection at single predefined concentrations of meglumine antimoniate and miltefosine and 50% effective doses (ED(50)s) were measured and correlated. The effects of 34°C and 37°C incubation temperatures and different parasite-to-host cell ratios on drug susceptibility were evaluated at 5, 10, and 20 parasites/cell. Reduction of the intracellular burden of Leishmania amastigotes in U-937 cells exposed to the predefined concentrations of meglumine antimoniate or miltefosine discriminated sensitive and experimentally derived resistant Leishmania populations and was significantly correlated with ED(50) values of clinical strains (for meglumine antimoniate, ρ = -0.926 and P < 0.001; for miltefosine, ρ = -0.906 and P < 0.001). Incubation at 37°C significantly inhibited parasite growth compared to that at 34°C in the absence of antileishmanial drugs and resulted in a significantly lower ED(50) in the presence of drugs. Susceptibility assessment was not altered by the parasite-to-cell ratio over the range evaluated. In conclusion, measurement of the reduction of parasite burden at a single predetermined drug concentration under standardized conditions provides an efficient and reliable strategy for susceptibility evaluation and monitoring of clinical strains of Leishmania.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JCM.00216-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3405580PMC
July 2012

Noninferiority of miltefosine versus meglumine antimoniate for cutaneous leishmaniasis in children.

J Infect Dis 2012 Feb 11;205(4):684-92. Epub 2012 Jan 11.

Centro Internacional De Entrenamiento E Investigaciones Médicas (CIDEIM), Cali, Colombia.

Background: Children have a lower response rate to antimonial drugs and higher elimination rate of antimony (Sb) than adults. Oral miltefosine has not been evaluated for pediatric cutaneous leishmaniasis.

Methods: A randomized, noninferiority clinical trial with masked evaluation was conducted at 3 locations in Colombia where Leishmania panamensis and Leishmania guyanensis predominated. One hundred sixteen children aged 2-12 years with parasitologically confirmed cutaneous leishmaniasis were randomized to directly observed treatment with meglumine antimoniate (20 mg Sb/kg/d for 20 days; intramuscular) (n = 58) or miltefosine (1.8-2.5 mg/kg/d for 28 days; by mouth) (n = 58). Primary outcome was treatment failure at or before week 26 after initiation of treatment. Miltefosine was noninferior if the proportion of treatment failures was ≤15% higher than achieved with meglumine antimoniate (1-sided test, α = .05).

Results: Ninety-five percent of children (111/116) completed follow-up evaluation. By intention-to-treat analysis, failure rate was 17.2% (98% confidence interval [CI], 5.7%-28.7%) for miltefosine and 31% (98% CI, 16.9%-45.2%) for meglumine antimoniate. The difference between treatment groups was 13.8%, (98% CI, -4.5% to 32%) (P = .04). Adverse events were mild for both treatments.

Conclusions: Miltefosine is noninferior to meglumine antimoniate for treatment of pediatric cutaneous leishmaniasis caused by Leishmania (Viannia) species. Advantages of oral administration and low toxicity favor use of miltefosine in children.

Clinical Trial Registration: NCT00487253.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/infdis/jir816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3266136PMC
February 2012

Toll-like receptors participate in macrophage activation and intracellular control of Leishmania (Viannia) panamensis.

Infect Immun 2011 Jul 25;79(7):2871-9. Epub 2011 Apr 25.

Centro Internacional de Entrenamiento e Investigaciones Medicas, Cali, Colombia.

Toll-like receptors (TLRs) play a central role in macrophage activation and control of parasitic infections. Their contribution to the outcome of Leishmania infection is just beginning to be deciphered. We examined the interaction of Leishmania panamensis with TLRs in the activation of host macrophages. L. panamensis infection resulted in upregulation of TLR1, TLR2, TLR3, and TLR4 expression and induced tumor necrosis factor alpha (TNF-α) secretion by human primary macrophages at comparable levels and kinetics to those of specific TLR ligands. The TLR dependence of the host cell response was substantiated by the absence of TNF-α production in MyD88/TRIF(-/-) murine bone marrow-derived macrophages and mouse macrophage cell lines in response to promastigotes and amastigotes. Systematic screening of TLR-deficient macrophages revealed that TNF-α production was completely abrogated in TLR4(-/-) macrophages, consistent with the increased intracellular parasite survival at early time points of infection. TNF-α secretion was significantly reduced in macrophages lacking endosomal TLRs but was unaltered by a lack of TLR2 or MD-2. Together, these findings support the participation of TLR4 and endosomal TLRs in the activation of host macrophages by L. panamensis and in the early control of infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/IAI.01388-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3191987PMC
July 2011

Viability and burden of Leishmania in extralesional sites during human dermal leishmaniasis.

PLoS Negl Trop Dis 2010 Sep 14;4(9). Epub 2010 Sep 14.

Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), Cali, Colombia.

Background: The clinical and epidemiological significance of Leishmania DNA in extralesional sites is obscured by uncertainty of whether the DNA derives from viable parasites. To examine dissemination of Leishmania during active disease and the potential participation of human infection in transmission, Leishmania 7SLRNA was exploited to establish viability and estimate parasite burden in extralesional sites of dermal leishmaniasis patients.

Methods: The feasibility of discriminating parasite viability by PCR of Leishmania 7SLRNA was evaluated in relation with luciferase activity of luc transfected intracellular amastigotes in dose-response assays of Glucantime cytotoxicity. Monocytes, tonsil swabs, aspirates of normal skin and lesions of 28 cutaneous and 2 mucocutaneous leishmaniasis patients were screened by kDNA amplification/Southern blot. Positive samples were analyzed by quantitative PCR of Leishmania 7SLRNA genes and transcripts.

Results: 7SLRNA amplification coincided with luciferase activity, confirming discrimination of parasite viability. Of 22 patients presenting kDNA in extralesional samples, Leishmania 7SLRNA genes or transcripts were detected in one or more kDNA positive samples in 100% and 73% of patients, respectively. Gene and transcript copy number amplified from extralesional tissues were comparable to lesions. 7SLRNA transcripts were detected in 13/19 (68%) monocyte samples, 5/12 (42%) tonsil swabs, 4/11 (36%) normal skin aspirates, and 22/25 (88%) lesions; genes were quantifiable in 15/19 (79%) monocyte samples, 12/13 (92%) tonsil swabs, 8/11 (73%) normal skin aspirates.

Conclusion: Viable parasites are present in extralesional sites, including blood monocytes, tonsils and normal skin of dermal leishmaniasis patients. Leishmania 7SLRNA is an informative target for clinical and epidemiologic investigations of human leishmaniasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0000819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2939031PMC
September 2010

T-bet, GATA-3, and Foxp3 expression and Th1/Th2 cytokine production in the clinical outcome of human infection with Leishmania (Viannia) species.

J Infect Dis 2010 Aug;202(3):406-15

Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), Cali, Colombia.

Background: T cell differentiation determines susceptibility and resistance to experimental cutaneous leishmaniasis, yet mixed T1/Th2 responses characterize the clinical spectrum of human infection with Leishmania (Viannia) species.

Materials And Methods: To discern the interrelationship of T cell differentiation and outcome of human infection, we examined factors that regulate T cell differentiation and Th1/Th2 cytokine responses in asymptomatic infection, active and historical chronic and recurrent cutaneous leishmaniasis. T-bet, GATA-3, Foxp3, and cytokine gene expression were quantified by real-time polymerase chain reaction and correlated with interleukin 2, interferon gamma, tumor necrosis factor alpha, interleukin 4, interleukin 13, and interleukin 10 secretion during in vitro response to live Leishmania panamensis.

Results: Higher GATA-3 expression than T-bet expression occurred throughout the 15 days of coculture with promastigotes; however, neither transcription nor secretion of interleukin 4 was detected. A sustained inverse correlation between GATA-3 expression and secretion of proinflammatory cytokines interferon gamma and tumor necrosis factor alpha was observed in asymptomatic infection. In contrast, higher T-bet expression and a higher ratio of T-bet to GATA-3 characterized active recurrent disease. Down-regulation of T-bet and GATA-3 expression and increased interleukin 2 secretion, compared with control subjects, was directly correlated with Foxp3 expression and interleukin 13 secretion in chronic disease.

Conclusions: Regulation of the inflammatory response rather than biased Th1/Th2 response distinguished asymptomatic and recalcitrant outcomes of infection with Leishmnania viannia species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1086/653829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850829PMC
August 2010