Consolidation of a Molecular Signature of Healing in Cutaneous Leishmaniasis Is Achieved during the First 10 Days of Treatment.

The immune response is central to the pathogenesis of cutaneous leishmaniasis (CL). However, most of our current understanding of the immune response in human CL derives from the analysis of systemic responses, which only partially reflect what occurs in the skin. In this study, we characterized the transcriptional dynamics of skin lesions during the course of treatment of CL patients and identified gene signatures and pathways associated with healing and nonhealing responses. We performed a comparative transcriptome profiling of serial skin lesion biopsies obtained before, in the middle, and at the end of treatment of CL patients (eight who were cured and eight with treatment failure). Lesion transcriptomes from patients who healed revealed recovery of the stratum corneum, suppression of the T cell-mediated inflammatory response, and damping of neutrophil activation, as early as 10 d after initiation of treatment. These transcriptional programs of healing were consolidated before lesion re-epithelization. In stark contrast, downregulation of genes involved in keratinization was observed throughout treatment in patients who did not heal, indicating that in addition to uncontrolled inflammation, treatment failure of CL is mediated by impaired mechanisms of wound healing. This work provides insights into the factors that contribute to the effective resolution of skin lesions caused by Leishmania (Viannia) species, sheds light on the consolidation of transcriptional programs of healing and nonhealing responses before the clinically apparent resolution of skin lesions, and identifies inflammatory and wound healing targets for host-directed therapies for CL.

Potency and Preclinical Evidence of Synergy of Oral Azole Drugs and Miltefosine in an Ex Vivo Model of Leishmania (Viannia) panamensis Infection.

Failure of treatment of cutaneous leishmaniasis with antimonial drugs and miltefosine is frequent. Use of oral combination therapy represents an attractive strategy to increase efficacy of treatment and reduce the risk of drug resistance. We evaluated the potency of posaconazole, itraconazole, voriconazole, and fluconazole and the potential synergy of those demonstrating the highest potency, in combination with miltefosine (HePC), against infection with Leishmania (Viannia) panamensis. Synergistic activity was determined by isobolograms and calculation of the fractional inhibitory concentration index (FICI), based on parasite quantification using an ex vivo model of human peripheral blood mononuclear cells (PBMCs) infected with a luciferase-transfected, antimony and miltefosine sensitive line of L. panamensis. The drug combination and concentrations that displayed synergy were then evaluated for antileishmanial effect in 10 clinical strains of L. panamensis by reverse transcription-quantitative (qRT-PCR) of Leishmania 7SLRNA. High potency was substantiated for posaconazole and itraconazole against sensitive as well as HePC- and antimony-resistant lines of L. panamensis, whereas fluconazole and voriconazole displayed low potency. HePC combined with posaconazole (Poz) demonstrated evidence of synergy at free drug concentrations achieved in plasma during treatment (2 µM HePC plus 4 µM Poz). FICI, based on 70% and 90% reduction of infection, was 0.5 for the sensitive line. The combination of 2 µM HePC plus 4 µM Poz effected a significantly greater reduction of infection by clinical strains of L. panamensis than individual drugs. Orally administrable miltefosine/posaconazole combinations demonstrated synergistic antileishmanial capacity ex vivo against L. panamensis, supporting their potential as a novel therapeutic strategy to improve efficacy and effectiveness of treatment.

Metabolomic Profiling of End-Stage Heart Failure Secondary to Chronic Chagas Cardiomyopathy.

Chronic Chagas cardiomyopathy (CCC) is the most frequent and severe clinical form of chronic Chagas disease, representing one of the leading causes of morbidity and mortality in Latin America, and a growing global public health problem. There is currently no approved treatment for CCC; however, omics technologies have enabled significant progress to be made in the search for new therapeutic targets. The metabolic alterations associated with pathogenic mechanisms of CCC and their relationship to cellular and immunopathogenic processes in cardiac tissue remain largely unknown. This exploratory study aimed to evaluate the potential underlying pathogenic mechanisms in the failing myocardium of patients with end-stage heart failure (ESHF) secondary to CCC by applying an untargeted metabolomic profiling approach. Cardiac tissue samples from the left ventricle of patients with ESHF of CCC etiology (n = 7) and healthy donors (n = 7) were analyzed using liquid chromatography-mass spectrometry. Metabolite profiles showed altered branched-chain amino acid and acylcarnitine levels, decreased fatty acid uptake and oxidation, increased activity of the pentose phosphate pathway, dysregulation of the TCA cycle, and alterations in critical cellular antioxidant systems. These findings suggest processes of energy deficit, alterations in substrate availability, and enhanced production of reactive oxygen species in the affected myocardium. This profile potentially contributes to the development and maintenance of a chronic inflammatory state that leads to progression and severity of CCC. Further studies involving larger sample sizes and comparisons with heart failure patients without CCC are needed to validate these results, opening an avenue to investigate new therapeutic approaches for the treatment and prevention of progression of this unique and severe cardiomyopathy.

Immuno-pharmacokinetics of Meglumine Antimoniate in Patients With Cutaneous Leishmaniasis Caused by Leishmania (Viannia).

BACKGROUND: Control of cutaneous leishmaniasis (CL) relies on chemotherapy, yet gaps in our understanding of the determinants of therapeutic outcome impede optimization of antileishmanial drug regimens. Pharmacodynamic (PD) parameters of antimicrobials are based on the relationship between drug concentrations/exposure and microbial kill. However, viable Leishmania persist in a high proportion of individuals despite clinical resolution, indicating that determinants other than parasite clearance are involved in drug efficacy. METHODS: In this study, the profiles of expression of neutrophils, monocytes, Th1 and Th17 gene signatures were characterized in peripheral blood mononuclear cells (PBMCs) during treatment with meglumine antimoniate (MA) and clinical cure of human CL caused by Leishmania (Viannia). We explored relationships of immune gene expression with plasma and intracellular antimony (Sb) concentrations. RESULTS: Our findings show a rapid and orchestrated modulation of gene expression networks upon exposure to MA. We report nonlinear pharmacokinetic/pharmacodynamic (PK/PD) relationships of Sb and gene expression dynamics in PBMCs , concurring with a time lag in the detection of intracellular drug concentrations and with PK evidence of intracellular Sb accumulation. CONCLUSIONS: Our results quantitatively portray the immune dynamics of therapeutic healing, and provide the knowledge base for optimization of antimonial drug treatments, guiding the selection and/or design of targeted drug delivery systems and strategies for targeted immunomodulation.

Immune Profile of the Nasal Mucosa in Patients with Cutaneous Leishmaniasis.

Localized skin lesions are characteristic of cutaneous leishmaniasis (CL); however, Leishmania (Viannia) species, which are responsible for most CL cases in the Americas, can spread systemically, sometimes resulting in mucosal disease. Detection of Leishmania has been documented in healthy mucosal tissues (conjunctiva, tonsils, and nasal mucosa) and healthy skin of CL patients and in individuals with asymptomatic infection in areas of endemicity of L (V) panamensis and L (V) braziliensis transmission. However, the conditions and mechanisms that favor parasite persistence in healthy mucosal tissues are unknown. In this descriptive study, we compared the cell populations of the nasal mucosa (NM) of healthy donors and patients with active CL and explored the immune gene expression signatures related to molecular detection of Leishmania in this tissue in the absence of clinical signs or symptoms of mucosal disease. The cellular composition and gene expression profiles of NM samples from active CL patients were similar to those of healthy volunteers, with a predominance of epithelial over immune cells, and within the CD45+ cell population, a higher frequency of CD66b+ followed by CD14+ and CD3+ cells. In CL patients with molecular evidence of Leishmania persistence in the NM, genes characteristic of an anti-inflammatory and tissue repair responses (IL4R, IL5RA, POSTN, and SATB1) were overexpressed relative to NM samples from CL patients in which Leishmania was not detected. Here, we report the first immunological description of subclinically infected NM tissues of CL patients and provide evidence of a local anti-inflammatory environment favoring parasite persistence in the NM.

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

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 Leishmania (Viannia) 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 ex vivo response of PBMCs to L (V) panamensis 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 (n = 8), compared to those from patients with treatment failure (n = 8). Among differentially expressed genes, pretreatment expression of CCL2 was significantly predictive of the therapeutic response (receiver operating characteristic [ROC] curve, area under the curve [AUC] = 0.82, P = 0.02). Polymorphisms in regulatory regions of the CCL2 promoter were analyzed in a pilot cohort of DNA samples from CL patients (cures, n = 20, and treatment failure, n = 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.

Phenotypic and functional stability of leukocytes from human peripheral blood samples: considerations for the design of immunological studies.

BACKGROUND: Human peripheral blood mononuclear cells (PBMCs) are extensively used for research of immune cell functions, identification of biomarkers and development of diagnostics and therapeutics for human diseases, among others. The assumption that "old blood samples" are not appropriate for isolation of PBMCs for functional assays has been a dogma in the scientific community. However, partial data on the impact of time after phlebotomy on the quality and stability of human PBMCs preparations impairs the design of studies in which time-controlled blood sampling is challenging such as field studies involving multiple sampling centers/sites. In this study, we evaluated the effect of time after phlebotomy over a 24 h time course, on the stability of human blood leukocytes used for immunological analyses. Blood samples from eight healthy adult volunteers were obtained and divided into four aliquots, each of which was left in gentle agitation at room temperature (24 °C) for 2 h (control), 7 h, 12 h and 24 h post phlebotomy. All samples at each time point were independently processed for quantification of mononuclear cell subpopulations, cellular viability, gene expression and cytokine secretion. RESULTS: A 24 h time delay in blood sample processing did not affect the viability of PBMCs. However, a significantly lower frequency of CD3+ T cells (p < 0.05) and increased LPS-induced CXCL10 secretion were observed at 12 h post-phlebotomy. Alterations in TNFα, CCL8, CCR2 and CXCL10 gene expression were found as early as 7 h after blood sample procurement. CONCLUSIONS: These data reveal previously unrecognized early time-points for sample processing control, and provide an assay-specific time reference for the design of studies that involve immunological analyses of human blood samples.

Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Assay for Diagnosis of Cutaneous and Visceral Leishmaniasis.

A novel pan-Leishmania loop-mediated isothermal amplification (LAMP) assay for the diagnosis of cutaneous and visceral leishmaniasis (CL and VL) that can be used in near-patient settings was developed. Primers were designed based on the 18S ribosomal DNA (rDNA) and the conserved region of minicircle kinetoplast DNA (kDNA), selected on the basis of high copy number. LAMP assays were evaluated for CL diagnosis in a prospective cohort trial of 105 patients in southwest Colombia. Lesion swab samples from CL suspects were collected and were tested using the LAMP assay, and the results were compared to those of a composite reference of microscopy and/or culture in order to calculate diagnostic accuracy. LAMP assays were tested on samples (including whole blood, peripheral blood mononuclear cells, and buffy coat) from 50 suspected VL patients from Ethiopia. Diagnostic accuracy was calculated against a reference standard of microscopy of splenic or bone marrow aspirates. To calculate analytical specificity, 100 clinical samples and isolates from fever-causing pathogens, including malaria parasites, arboviruses, and bacteria, were tested. We found that the LAMP assay had a sensitivity of 95% (95% confidence interval [CI], 87.2% to 98.5%) and a specificity of 86% (95% CI, 67.3% to 95.9%) for the diagnosis of CL. With VL suspects, the sensitivity of the LAMP assay was 92% (95% CI, 74.9% to 99.1%) and its specificity was 100% (95% CI, 85.8% to 100%) in whole blood. For CL, the LAMP assay is a sensitive tool for diagnosis and requires less equipment, time, and expertise than alternative CL diagnostics. For VL, the LAMP assay using a minimally invasive sample is more sensitive than the gold standard. Analytical specificity was 100%.

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

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.

Functional Validation of ABCA3 as a Miltefosine Transporter in Human Macrophages: IMPACT ON INTRACELLULAR SURVIVAL OF LEISHMANIA (VIANNIA) PANAMENSIS.

Within its mammalian host, Leishmania resides and replicates as an intracellular parasite. The direct activity of antileishmanials must therefore depend on intracellular drug transport, metabolism, and accumulation within the host cell. In this study, we explored the role of human macrophage transporters in the intracellular accumulation and antileishmanial activity of miltefosine (MLF), the only oral drug available for the treatment of visceral and cutaneous leishmaniasis (CL). Membrane transporter gene expression in primary human macrophages infected in vitro with Leishmania Viannia panamensis and exposed to MLF showed modulation of ABC and solute liquid carrier transporters gene transcripts. Among these, ABCA3, a lipid transporter, was significantly induced after exposure to MLF, and this induction was confirmed in primary macrophages from CL patients. Functional validation of MLF as a substrate for ABCA3 was performed by shRNA gene knockdown (KD) in THP-1 monocytes. Intracellular accumulation of radiolabeled MLF was significantly higher in ABCA3(KD) macrophages. ABCA3(KD) resulted in increased cytotoxicity induced by MLF exposure. ABCA3 gene expression inversely correlated with intracellular MLF content in primary macrophages from CL patients. ABCA3(KD) reduced parasite survival during macrophage infection with an L. V. panamensis strain exhibiting low in vitro susceptibility to MLF. Confocal microscopy showed ABCA3 to be located in the cell membrane of resting macrophages and in intracellular compartments in L. V. panamensis-infected cells. These results provide evidence of ABCA3 as an MLF efflux transporter in human macrophages and support its role in the direct antileishmanial effect of this alkylphosphocholine drug.

Pharmacokinetics of Miltefosine in Children and Adults with Cutaneous Leishmaniasis.

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. Leishmania (Viannia) panamensis 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 (P < 0.01). Leishmania 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. In vitro miltefosine susceptibility was similar for Leishmania 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.).

Screening of TNFα, IL-10 and TLR4 single nucleotide polymorphisms in individuals with asymptomatic and chronic cutaneous leishmaniasis in Colombia: a pilot study.

BACKGROUND: Clinical manifestations of cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) range from asymptomatic infection to self-limited, or chronic (non-healing) cutaneous lesions. Given the critical role of the immune response in the clinical outcome of CL, it is plausible that functional polymorphisms in immune-related genes contribute to define the clinical manifestations of human infection. METHODS: DNA samples from a retrospective cohort of individuals from an endemic area of L. V. panamensis transmission in Colombia were used to determine the frequency of SNPs in TNFα, IL-10 and TLR4 genes. DNA samples were obtained from 74 adult participants: 38 patients presenting chronic cutaneous leishmaniasis (CCL) and 36 individuals with asymptomatic infection. Genotyping of TNFα-308G/A, IL-10-819C/T, and TLR4 Asp299Gly and Thr399Ile SNPs, was conducted by PCR-restriction fragment length polymorphisms. Allele, genotype frequencies and associations between SNPs and clinical groups were evaluated. RESULTS: The A allele in TNFα-308G/A SNP was found more frequently in individuals with asymptomatic infection (16% vs 7%), whereas the CC genotype in IL-10-819 C/T SNP was more frequent in patients with CCL (34% vs. 27% in asymptomatic individuals). No differences in allele frequencies for TLR4 SNPs were found among groups. CONCLUSION: This study provides a reference base for statistical power calculation and design of association studies of genetic polymorphisms in immune response related-genes and the pathogenesis of infections caused by L. V. panamensis.

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

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.

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

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/ß-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.

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

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.

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

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.

Innate biosignature of treatment failure in human cutaneous leishmaniasis.

The quality and magnitude of the immune and inflammatory responses determine the clinical outcome of Leishmania infection, and contribute to the efficacy of antileishmanial treatments. However, the precise immune mechanisms involved in healing or in chronic immunopathology of human cutaneous leishmaniasis (CL) are not completely understood. Through sequential transcriptomic profiling of blood monocytes (Mo), neutrophils (Nφ), and eosinophils (Eφ) over the course of systemic treatment with meglumine antimoniate, we discovered that a heightened and sustained Type I interferon (IFN) response signature is a hallmark of treatment failure (TF) in CL patients. The transcriptomes of pre-treatment, mid-treatment and end-of-treatment samples were interrogated to identify predictive and prognostic biomarkers of TF. A composite score derived from the expression of 9 differentially expressed genes (common between Mo, Nφ and Eφ) was predictive of TF in this patient cohort for biomarker discovery. Similarly, machine learning models constructed using data from pre-treatment as well as post-treatment samples, accurately classified treatment outcome between cure and TF. Results from this study instigate the evaluation of Type-I IFN responses as new immunological targets for host-directed therapies for treatment of CL, and highlight the feasibility of using transcriptional signatures as predictive biomarkers of outcome for therapeutic decision making.

Leishmania RNA Virus Is Not Detected in All Species of the Leishmania Viannia Subgenus: The Case of L. (V.) panamensis in Colombia.

The endosymbiotic Leishmania RNA virus 1 (LRV1) has been associated with severity and clinical manifestations of American tegumentary leishmaniasis caused by species of the Leishmania (Viannia) subgenus. Between and within Leishmania species, and among endemic countries, the prevalence of LRV is highly variable. The LRV virus has not been detected in L. (V.) panamensis, the second-most prevalent species in Central America and Colombia. However, no systematic screening of LRV has been conducted in L. (V.) panamensis, and thus it is still controversial whether this virus is truly absent from the species. We sought to determine the prevalence of LRV1 in L. (V.) panamensis clinical strains isolated from patients with cutaneous leishmaniasis (CL), from different geographic areas of Colombia. We analyzed 219 clinical strains; 78% were L. (V.) panamensis, 18% were L. (V.) braziliensis, and 4% were L. (V.) guyanensis. Screening for LRV1 was performed by quantitative reverse transcription-polymerase chain reaction. The LRV1 was detected in 18% (7 of 40) of L. (V) braziliensis strains, and was not detected in any of the L. (V.) guyanensis or L. (V.) panamensis strains. The LRV1-positive L. (V). braziliensis strains came from the Amazon Basin. Of the seven LRV1-positive strains, two were isolated from patients with mucocutaneous leishmaniasis, and the remaining from patients with CL. Our results confirm the absence of LRV1 in L. (V.) panamensis in Colombia.

Macrophage metallothioneins participate in the antileishmanial activity of antimonials.

Host cell functions that participate in the pharmacokinetics and pharmacodynamics (PK/PD) of drugs against intracellular pathogen infections are critical for drug efficacy. In this study, we investigated whether macrophage mechanisms of xenobiotic detoxification contribute to the elimination of intracellular Leishmania upon exposure to pentavalent antimonials (SbV). Primary macrophages from patients with cutaneous leishmaniasis (CL) (n=6) were exposed ex vivo to L. V. panamensis infection and SbV, and transcriptomes were generated. Seven metallothionein (MT) genes, potent scavengers of heavy metals and central elements of the mammalian cell machinery for xenobiotic detoxification, were within the top 20 up-regulated genes. To functionally validate the participation of MTs in drug-mediated killing of intracellular Leishmania, tandem knockdown (KD) of MT2-A and MT1-E, MT1-F, and MT1-X was performed using a pan-MT shRNA approach in THP-1 cells. Parasite survival was unaffected in tandem-KD cells, as a consequence of strong transcriptional upregulation of MTs by infection and SbV, overcoming the KD effect. Gene silencing of the metal transcription factor-1 (MTF-1) abrogated expression of MT1 and MT2-A genes, but not ZnT-1. Upon exposure to SbV, intracellular survival of Leishmania in MTF-1KD cells was significantly enhanced. Results from this study highlight the participation of macrophage MTs in Sb-dependent parasite killing.

Leishmania-induced inactivation of the macrophage transcription factor AP-1 is mediated by the parasite metalloprotease GP63.

Leishmania parasites have evolved sophisticated mechanisms to subvert macrophage immune responses by altering the host cell signal transduction machinery, including inhibition of JAK/STAT signalling and other transcription factors such as AP-1, CREB and NF-κB. AP-1 regulates pro-inflammatory cytokines, chemokines and nitric oxide production. Herein we show that upon Leishmania infection, AP-1 activity within host cells is abolished and correlates with lower expression of 5 of the 7 AP-1 subunits. Of interest, c-Jun, the central component of AP-1, is cleaved by Leishmania. Furthermore, the cleavage of c-Jun is dependent on the expression and activity of the major Leishmania surface protease GP63. Immunoprecipitation of c-Jun from nuclear extracts showed that GP63 interacts, and cleaves c-Jun at the perinuclear area shortly after infection. Phagocytosis inhibition by cytochalasin D did not block c-Jun down-regulation, suggesting that internalization of the parasite might not be necessary to deliver GP63 molecules inside the host cell. This observation was corroborated by the maintenance of c-Jun cleavage upon incubation with L. mexicana culture supernatant, suggesting that secreted, soluble GP63 could use a phagocytosis-independent mechanism to enter the host cell. In support of this, disruption of macrophage lipid raft microdomains by Methyl ß-Cyclodextrin (MßCD) partially inhibits the degradation of full length c-Jun. Together our results indicate a novel role of the surface protease GP63 in the Leishmania-mediated subversion of host AP-1 activity.