Natural resistance to meglumine antimoniate is associated with treatment failure in cutaneous leishmaniasis caused by Leishmania (Viannia) panamensis.

The multifactorial basis of therapeutic response can obscure the relation between antimicrobial drug susceptibility and clinical outcome. To discern the relationship between parasite susceptibility to meglumine antimoniate (SbV) and therapeutic outcome of cutaneous leishmaniasis, risk factors for treatment failure were considered in evaluating this relationship in ninety-one cutaneous leishmaniasis patients and corresponding clinical strains of Leishmania (Viannia) panamensis. Parasite susceptibility to 32 µg SbV/mL (plasma Cmax) was evaluated in primary human macrophages, PBMCs, and U937 macrophages. Early parasitological response to treatment was determined in lesions of a subgroup of patients, and pathogenicity of Sb-resistant and sensitive clinical strains was compared in BALB/c mice. Parasite survival in cell models and patient lesions was determined by qRT-PCR of Leishmania 7SLRNA transcript. Parasite loads in BALB/c mice were quantified by limiting dilution analysis. The disparate Sb-susceptibility of parasite subpopulations distinguished by isoenzyme profiles (zymodemes) was manifest in all cell models. Notably, Sb-resistance defined by parasite survival, was most effectively discerned in U937 macrophages compared with primary human host cells, significantly higher among strains from patients who failed treatment than cured and, significantly associated with treatment failure. Each unit increase in transformed survival rate corresponded to a 10.6-fold rise in the odds of treatment failure. Furthermore, treatment failure was significantly associated with naturally Sb-resistant zymodeme 2.3 strains, which also produced larger lesions and parasite burdens in BALB/c mice than Sb-sensitive zymodeme 2.2 strains. The confounding effect of host risk factors for treatment failure in discerning this association was evidenced in comparing strains from patients with and without the defined risk factors for treatment failure. These results establish the association of natural resistance to meglumine antimoniate with treatment failure, the importance of host risk factors in evaluating drug susceptibility and treatment outcome, and the clinical and epidemiological relevance of natural Sb-resistance in L. (V.) panamensis subpopulations.

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.

Sequencing of hsp70 for discernment of species from the Leishmania (Viannia) guyanensis complex from endemic areas in Colombia.

BACKGROUND: Colombia is ranked very high among countries with the highest numbers of endemic Leishmania species (n = 9) causing human disease. Although much effort has been devoted to generating simple and specific tools for Leishmania species identification, challenges remain in the discrimination of species belonging to the Leishmania (Viannia) guyanensis complex: L. (V.) guyanensis and L. (V.) panamensis. METHODS: A set of seven reference strains of species belonging to the L. (Leishmania) and L. (Viannia) subgenera, clinical strains from human cases of cutaneous leishmaniasis (CL; n = 26) and samples collected from sylvatic mammals and sand flies (n = 7) from endemic areas in Colombia were analyzed in this study. The heat-shock protein 70 gene (hsp70) was amplified by PCR from DNA extracted from logarithmic-phase promastigotes or tissue samples, and the PCR products were sequenced. Sequence alignment was performed against a set of previously published and curated sequences, and phylogenetic analysis based on the maximum-likelihood and Bayesian inference approaches was conducted. Haplotype diversity among strains and species of the L. (V.) guyanensis complex was explored using a median-joining network. RESULTS: Sequencing of the hsp70 gene for L. (Viannia) spp. typing was comparable to species identification using isoenzyme electrophoresis or monoclonal antibodies. Complete species matching was found, except for one sylvatic sample with an identity yet unsolved. Among the L. (V.) panamensis clinical strains, two distinctive phylogenetic clusters were found to correlate with two different zymodemes: L. (V.) panamensis Z2.2 and Z2.3. Analysis of samples from sylvatic environments identified novel records of naturally infected wild mammal and sand fly species. CONCLUSIONS: Our results support the adequacy of hsp70 gene sequencing as a single-locus approach for discrimination of L. (Viannia) spp., as well as for exploring the genetic diversity within the L. (V.) guyanensis complex.

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.

Early Leukocyte Responses in Ex-Vivo Models of Healing and Non-Healing Human Leishmania (Viannia) panamensis Infections.

Early host-pathogen interactions drive the host response and shape the outcome of natural infections caused by intracellular microorganisms. These interactions involve a number of immune and non-immune cells and tissues, along with an assortment of host and pathogen-derived molecules. Our current knowledge has been predominantly derived from research on the relationships between the pathogens and the invaded host cell(s), limiting our understanding of how microbes elicit and modulate immunological responses at the organismal level. In this study, we explored the early host determinants of healing and non-healing responses in human cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) panamensis. We performed a comparative transcriptomic profiling of peripheral blood mononuclear cells from healthy donors (PBMCs, n=3) exposed to promastigotes isolated from patients with chronic (CHR, n=3) or self-healing (SH, n=3) CL, and compared these to human macrophage responses. Transcriptomes of L. V. panamensis-infected PBMCs showed enrichment of functional gene categories derived from innate as well as adaptive immune cells signatures, demonstrating that Leishmania modulates adaptive immune cell functions as early as after 24h post interaction with PBMCs from previously unexposed healthy individuals. Among differentially expressed PBMC genes, four broad categories were commonly modulated by SH and CHR strains: cell cycle/proliferation/differentiation, metabolism of macromolecules, immune signaling and vesicle trafficking/transport; the first two were predominantly downregulated, and the latter upregulated in SH and CHR as compared to uninfected samples. Type I IFN signaling genes were uniquely up-regulated in PBMCs infected with CHR strains, while genes involved in the immunological synapse were uniquely downregulated in SH infections. Similarly, pro-inflammatory response genes were upregulated in isolated macrophages infected with CHR strains. Our data demonstrate that early responses during Leishmania infection extend beyond innate cell and/or phagocytic host cell functions, opening new frontiers in our understanding of the triggers and drivers of human CL.

Comparative Assessment of DNA Targets and Amplification Methods for Leishmania (Viannia) Detection in Human Samples.

Multiple polymerase chain reaction (PCR)-based approaches have been developed for Leishmania detection in clinical and laboratory samples, and this diversity limits inter-study comparisons, meta-analyses, and generalization of findings. Towards harmonization of a molecular tool for detection of Leishmania (Viannia) for research purposes, we evaluated the concordance of 18SrDNA quantitative polymerase chain reaction (qPCR) and minicircle kinetoplastid DNA (mkDNA) PCR followed by Southern blot (PCR-SB) in in vitro infection systems and in lesion and mucosal swab samples from Colombian patients with cutaneous leishmaniasis caused by L. (Viannia). The lower limit of parasite detection of 18SrDNA qPCR and mkDNA PCR-SB was 10-1 promastigotes and one intracellular amastigote per reaction. From cutaneous lesions (n = 63), an almost perfect concordance was found between the methods (κ = 0.92, 95% CI: 0.82-1.00). Despite equal limits of detection, mkDNA PCR-SB was more efficient for parasite detection in mucosal samples than 18SrDNA qPCR or 18SrDNA digital droplet PCR. The high concordance, sensitivity, scaling potential, and feasibility of implementation of the 18SrDNA qPCR, support its selection as the L. (Viannia) in research laboratories, as a first step towards harmonization of research protocols in the region.

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.

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

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.

Parasitological Confirmation and Analysis of Leishmania Diversity in Asymptomatic and Subclinical Infection following Resolution of Cutaneous Leishmaniasis.

BACKGROUND: The contribution of individuals with subclinical infection to the transmission and endemicity of cutaneous leishmaniasis (CL) is unknown. Immunological evidence of exposure to Leishmania in residents of endemic areas has been the basis for defining the human population with asymptomatic infection. However, parasitological confirmation of subclinical infection is lacking. METHODS: We investigated the presence and viability of Leishmania in blood and non-invasive mucosal tissue samples from individuals with immunological evidence of subclinical infection in endemic areas for CL caused by Leishmania (Viannia) in Colombia. Detection of Leishmania kDNA was conducted by PCR-Southern Blot, and parasite viability was confirmed by amplification of parasite 7SLRNA gene transcripts. A molecular tool for genetic diversity analysis of parasite populations causing persistent subclinical infection based on PCR amplification and sequence analysis of an 82bp region between kDNA conserved blocks 1 and 2 was developed. PRINCIPAL FINDINGS: Persistent Leishmania infection was demonstrated in 40% (46 of 114) of leishmanin skin test (LST) positive individuals without active disease; parasite viability was established in 59% of these (27 of 46; 24% of total). Parasite burden quantified from circulating blood monocytes, nasal, conjunctival or tonsil mucosal swab samples was comparable, and ranged between 0.2 to 22 parasites per reaction. kDNA sequences were obtained from samples from 2 individuals with asymptomatic infection and from 26 with history of CL, allowing genetic distance analysis that revealed diversity among sequences and clustering within the L. (Viannia) subgenus. CONCLUSIONS: Our results provide parasitological confirmation of persistent infection among residents of endemic areas of L. (Viannia) transmission who have experienced asymptomatic infection or recovered from CL, revealing a reservoir of infection that potentially contributes to the endemicity and transmission of disease. kDNA genotyping establishes proof-of-principle of the feasibility of genetic diversity analysis in previously inaccessible and unexplored parasite populations in subclinically infected individuals.

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

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".