Molecular typing of Leishmania (Leishmania) amazonensis and species of the subgenus Viannia associated with cutaneous and mucosal leishmaniasis in Colombia: A concordance study

Introduction: Multilocus enzyme electrophoresis (MLEE) is the reference standard for the characterization of Leishmania species. The test is restricted to specialized laboratories due to its technical complexity, cost, and time required to obtain results. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is used to identify Leishmania species. Objective: To establish the concordance between the two tests as identifying methods for circulating species in Colombia. Materials and methods: A total of 96 isolates from patients with cutaneous or mucosal leishmaniasis were selected and identified by MLEE and PCR-RFLP with miniexon and hsp70 as the molecular targets, which were used sequentially. Restriction enzymes HaeIII and BccI were similarly applied. Cohen’s kappa coefficient and the 95% confidence interval (CI) were calculated. Results: The kappa coefficient and the 95% CI between MLEE and PCR-RFLP displayed “very good” concordance with a coefficient of 0.98 (CI95%: 0.98 to 1.00). The identified species were Leishmania Viannia braziliensis, Leishmania Viannia panamensis, Leishmania Viannia guyanensis and Leishmania Leishmania amazonensis. A total of 80 of the 96 isolates were sequenced and the results obtained by PCR-RFLP were confirmed. Conclusion: Due to the concordance obtained between tests results with the amplification of the genes miniexon and hsp70, PCR-RFLP is proposed as an alternative for identifying circulating Leishmania species in Colombia

Changes to cholesterol trafficking in macrophages by Leishmania parasites infection

Leishmania spp. are protozoan parasites that are transmitted by sandfly vectors during blood sucking to vertebrate hosts and cause a spectrum of diseases called leishmaniases. It has been demonstrated that host cholesterol plays an important role during Leishmania infection. Nevertheless, little is known about the intracellular distribution of this lipid early after internalization of the parasite. Here, pulse‐chase experiments with radiolabeled cholesteryl esterified to fatty acids bound to low‐density lipoproteins indicated that retention of this source of cholesterol is increased in parasite‐containing subcellular fractions, while uptake is unaffected. This is correlated with a reduction or absence of detectable NPC1 (Niemann–Pick disease, type C1), a protein responsible for cholesterol efflux from endocytic compartments, in the Leishmania mexicana habitat and infected cells. Filipin staining revealed a halo around parasites within parasitophorous vacuoles (PV) likely representing free cholesterol accumulation. Labeling of host cell membranous cholesterol by fluorescent cholesterol species before infection revealed that this pool is also trafficked to the PV but becomes incorporated into the parasites’ membranes and seems not to contribute to the halo detected by filipin. This cholesterol sequestration happened early after infection and was functionally significant as it correlated with the upregulation of mRNA‐encoding proteins required for cholesterol biosynthesis. Thus, sequestration of cholesterol by Leishmania amastigotes early after infection provides a basis to understand perturbation of cholesterol‐dependent processes in macrophages that were shown previously by others to be necessary for their proper function in innate and adaptive immune responses

Caracterización de un modelo celular diseñado para el tamizaje genético mediante RNA de interferencia en el complejo macrófago – infectado por Leishmania (Viannia) braziliensis

Las leishmaniasis son un grupo de enfermedades causadas por diferentes especies de parásitos del género Leishmania. En el desarrollo de la enfermedad el establecimiento de la infección es obligatorio y se da en parte, por la supervivencia y replicación del parásito en el macrófago. Por lo anterior es importante estudiar los mecanismos moleculares implicados en la interacción macrófago-Leishmania. La mayoría de los trabajos realizados sobre el efecto de la infección de Leishmania en la expresión de genes del macrófago se ha desarrollado con especies del subgénero Leishmania y en momentos iniciales de la infección. El objetivo de este trabajo fue identificar en la etapa del establecimiento de la infección, los genes y los procesos biológicos del macrófago potencialmente importantes en la interacción macrófago - L. braziliensis y caracterizar un modelo celular que permita la evaluación funcional de los genes identificados. El perfil de expresión de genes diferencialmente expresados entre macrófagos no infectados e infectados, se determinó por ensayos de microarreglos y los procesos biológicos por análisis de enriquecimiento funcional. Se evaluó el modelo celular: macrófagos derivados de la línea celular U937, en términos de su capacidad para ser modificado genéticamente, permitir el silenciamiento de genes por RNA de interferencia (shRNA) y permitir la identificación de cambios fenotípicos de infección, causados por dicho silenciamiento genético. Para lo anterior se expresó y silenció el gen gfp y se silenciaron los genes lmna y gro-β. El silenciamiento de los genes fue evaluado midiendo los niveles de expresión de la proteína por western blot y el efecto del silenciamiento, evaluando el fenotipo de infección de los macrófagos en términos de carga parasitaria y porcentaje de macrófagos infectados en las líneas silenciadas y en las líneas control. El proceso de silenciamiento fue exitoso para los tres genes estudiados obteniendo reducción del 88.9% en los niveles de GFP, del 87,5% en los niveles de LMNA y del 74,4% para Gro-β. El modelo celular permitió evidenciar cambios en la susceptibilidad de la infección de los macrófagos infectados por L. braziliensis, como consecuencia del silenciamiento genético. Los ensayos de microarreglos permitieron identificar 218 genes diferencialmente expresados entre macrófagos no infectados e infectados con L. (V.) braziliensis, y con base en los ensayos de enriquecimiento funcional se determinó que la biosíntesis de esteroles, específicamente la del colesterol era el principal proceso asociado. Los genes asociados a la biosíntesis del colesterol estaban regulados negativamente. Este es el primer estudio que reporta el perfil de expresión génica de macrófagos infectados por L. (V.) braziliensis y el proceso biológico principalmente asociado durante el establecimiento de la infección.Abstrat. The leishmaniasis are a group of diseases caused by different species of parasites of the genus Leishmania. In the development of the disease the establishment of infection is mandatory and is given partially by the survival and replication of the parasite in the macrophage. Therefore, it is important to study the molecular mechanisms involved in the interaction of macrophage-leishmania. Most of the work carried out on the effect of Leishmania infection in macrophage gene expression has been developed with species of the subgenus Leishmania and early moments of infection. The objective of this study was to identify, in the time of the establishment of infection, the macrophage´s genes and biological processes potentially important in the interaction macrophage - L. (V.) braziliensis; and characterize a cellular model that allows the functional evaluation of the genes identified. The expression profile of genes differentially expressed between macrophages uninfected and infected was determined by microarrays assays and biological processes by performance of functional enrichment analysis. The cellular model was assessed in terms of in terms of its ability to be genetically modified, if it allows gene silencing by RNA interference (shRNA) and if it allows the identification of phenotypic changes of infection, caused by gene silencing. In order to evaluate the parameters mentioned above, expression and silencing of gfp gene was performed as well as silencing of lmna and gro-β genes. The silencing of these genes was assessed by measuring the levels of protein expression by western blot and the effect of silencing process was evaluated identifying the phenotype of infection of macrophages in terms of parasite load and percentage of macrophages infected in silenced lines and control lines. The silencing process was successful for the three genes studied to obtaining reduction of 88.9% in levels of GFP, 87.5% in levels of LMNA and 74.4% to Gro-β. The cell model allowed identification of changes in the susceptibility of infection of macrophages infected by L. (V.) braziliensis, as a result of the gene silencing. The microarray assays helped identify 218 differentially expressed genes between macrophages uninfected and infected with L. (V.) braziliensis. Functional enrichment analysis determined that the biosynthesis of sterols, specifically the cholesterol, was the main associated process. The genes associated with cholesterol biosynthesis were downregulated. This is the first study that reports the profile of gene expression in macrophages infected with L. (V.) braziliensis and the main biological process associated during the establishment of the infection.Doctorad

Leishmaniasis mucosa: una enfermedad olvidada, descripción e identificación de especies en 50 casos colombianos

Introduction: Mucosal leishmaniasis has a progressive course and can cause deformity and even mutilation in the affected areas. It is endemic in the American continent and it is mainly caused by Leishmania (Viannia) braziliensis.Objective: To describe a series of mucosal leishmaniasis cases and the infectious Leishmania species.Materials and methods: We included 50 patients with a clinical diagnosis of mucosal leishmaniasis and parasitological confirmation, and we described their clinical and laboratory results. We performed species typing by PCR-RFLP using the miniexon sequence and hsp70 genes; confirmation was done by sequencing.Results: The median time of disease evolution was 2.9 years (range: 1 month to 16 years). The relevant clinical findings included mucosal infiltration (94%), cutaneous leishmaniasis scar (74%), total loss of the nasal septum (24%), nasal deformity (22%), and mucosal ulceration (38%). The symptoms reported included nasal obstruction (90%), epistaxis (72%), rhinorrhea (72%), dysphonia (28%), dysphagia (18%), and nasal pruritus (34%). The histopathological study revealed a pattern compatible with leishmaniasis in 86% of the biopsies, and amastigotes were identified in 14% of them. The Montenegro skin test was positive in 86% of patients, immunofluorescence in 84%, and culture in 8%. Leishmania (V.) braziliensis was identified in 88% of the samples, L. (V) panamensis in 8%, and L. (V.) guyanensis and L. (L.) amazonensis in 2% respectively.Conclusion: In this study, we found a severe nasal disease with destruction and deformity of the nasal septum in 25% of the cases, probably associated with late diagnosis. Leishmania (V.) braziliensis was the predominant species. We described a case of mucosal leishmaniasis in Colombia caused by L. (L.) amazonensis for the first time.Introducción. La leishmaniasis mucosa tiene un curso progresivo y puede causar deformidad e incluso mutilación de las zonas afectadas. Es endémica en el continente americano y es causada principalmente por Leishmania (Viannia) brasiliensis.Objetivo. Describir una serie de casos de leishmaniasis mucosa y las especies de Leishmania infecciosas.Materiales y métodos. Se estudiaron 50 pacientes con diagnóstico clínico de leishmaniasis mucosa y confirmación parasitológica. Se describieron sus características clínicas y los resultados de laboratorio. La tipificación de especies se hizo mediante reacción en cadena de la polimerasa de los polimorfismos de la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism Polymerase Chain Reaction, PCR-RFLP) en la secuencia del miniexon y el gen hsp70 y se confirmó por secuenciación.Resultados. La evolución de la enfermedad fue de un mes a dieciséis años (mediana de 2,8 años). Los hallazgos clínicos fueron los siguientes: infiltración mucosa (94 %), cicatriz de leishmaniasis cutánea (74 %), pérdida total del tabique nasal (24 %), deformidad nasal (22 %) y ulceración (38 %). Los síntomas reportados fueron: obstrucción nasal (90 %), epistaxis (72 %), rinorrea (72 %), disfonía (28 %), disfagia (18 %) y prurito nasal (34 %). La histopatología mostró un patrón compatible con leishmaniasis en 86 % de las biopsias y se identificaron amastigotes en 14 % de ellas. La prueba de Montenegro fue positiva en 86 % de los pacientes, la inmunofluorescencia en 84 %, y el cultivo en 8 %. Leishmania (V.) brasiliensis se identificó en 88 % de las muestras, L. (V) panamensis en 8 %, y L. (V.) guyanensis y L. (L.) amazonensis en 2 %, respectivamente.Conclusión. Se encontró enfermedad nasal grave con destrucción y deformidad del tabique nasal en una cuarta parte de los casos, probablemente debido a un diagnóstico tardío. Leishmania (V.) brasiliensis fue la especie predominante. Se describe por primera vez un caso de leishmaniasis mucosa causado por L. (L.) amazonensis en Colombia

Evaluating the spatial distribution of Leishmania parasites in Colombia from clinical samples and human isolates (1999 to 2016).

In Colombia, nine species of parasites of the genus Leishmania circulate in more than 20 sand fly species, putting at risk of contracting the disease approximately 60% of the population. The Federico Lleras Acosta Dermatological Center, a reference center in Colombia, has been treating patients with cutaneous and mucosal leishmaniasis for more than 15 years, identifying the infecting Leishmania species from different clinical samples, and recording systematically all the epidemiological and geographic information related to each diagnosed patient. With this valuable information, the objective of this work was to perform a long term and large-scale study, aiming to identify the Leishmania species circulating in Colombia from clinical samples from 1999 to 2016, and to assess their current and potential spatial distribution. In all, four Leishmania species were identified in 688 samples from 183 municipalities distributed in 28 of the 32 departments of the country, and 387 records were georeferenced, from 20 departments. The most widespread species was L. (V.) braziliensis, showing new collection records, and the species related to areas with highest leishmaniasis transmission was L. (V.) panamensis. Ecological niche models were built for the three species that had more than 20 georeferenced records, showing a potential distribution for L. (V.) braziliensis on 42% of the national territory mainly in the interandean valleys, and the Orinoquia and Amazon regions. Leishmania (V.) guyanensis potential distribution covers 36% of Colombia continental territory with a spatial distribution similar to that of L. (V.) braziliensis. There was a marked tendency of L. (V.) panamensis to be distributed in the northwest of the country occupying 35% of the national area and mainly in areas of transformed ecosystems. Species were identified in patients from areas where the occurrence of cases was unprecedented, which suggests that the distribution of Leishmania may be greater than currently known. To improve the predictive capacity of the models, we suggest incorporating, in future studies, Leishmania samples from vectors and reservoirs that have a greater dependence on environmental variables. Our results are an important tool for health systems because they allow potential areas of transmission and information gaps to be identified

Polymerase chain reaction with two molecular targets in mucosal leishmaniasis' diagnosis: a validation study

We validated the polymerase chain reaction (PCR) with a composite reference standard in 61 patients clinically suspected of having mucosal leishmaniasis, 36 of which were cases and 25 non-cases according to this reference standard. Patient classification and test application were carried out independently by two blind observers. One pair of primers was used to amplify a fragment of 120 bp in the conserved region of kDNA and another pair was used to amplify the internal transcript spacers (ITS) rDNA. PCR showed 68.6% (95% CI 59.2-72.6) sensitivity and 92% (95% CI 78.9-97.7) specificity; positive likelihood ratio: 8.6 (95% CI 2.8-31.3) and negative likelihood ratio: 0.3 (95% CI 0.3-0.5), when kDNA molecular target was amplified. The test performed better on sensitivity using this target compared to the ITS rDNA molecular target which showed 40% (95% CI 31.5-42.3) sensitivity and 96% (95% CI 84.1-99.3) specificity; positive likelihood ratio: 10 (95% CI 2.0-58.8) and negative likelihood ratio: 0.6 (95% CI 0.6-0.8). The inter-observer agreement was excellent for both tests. Based upon results obtained and due to low performance of conventional methods for diagnosing mucosal leishmaniasis, we consider PCR with kDNA as molecular target is a useful diagnostic test and the ITS rDNA molecular target is useful when the aim is to identify species

<i>In vitro</i> susceptibility of clinical isolates of <i>Leishmania Viannia</i> to AmB by species.

<p>A) Distribution of the mean effective concentration (EC₅₀) in μg/ml AmB for the strains according to species. B) Distribution of the percent reduction in the number of parasites at a concentration of 0.5 μg/ml AmB for the strains according to species.</p

Intraspecies differences in natural susceptibility to amphotericine B of clinical isolates of <i>Leishmania</i> subgenus <i>Viannia</i>

<div><p>Amphotericin B (AmB) is a recommended medication for the treatment of cutaneous and mucosal leishmaniasis in cases of therapeutic failure with first-line medications; however, little is known about the <i>in vitro</i> susceptibility to AmB of clinical isolates of the subgenus <i>Viannia</i>, which is most prevalent in South America. This work aimed to determine the <i>in vitro</i> susceptibility profiles to AmB of clinical isolates of the species <i>L</i>. <i>(V</i>.<i>) panamensis</i>, <i>L</i>. <i>(V</i>.<i>) guyanensis and L</i>. <i>(V</i>.<i>) braziliensis</i>. <i>In vitro</i> susceptibility to AmB was evaluated for 65 isolates. Macrophages derived from the U937 cell line were infected with promastigotes and exposed to different AmB concentrations. After 96 hours, the number of intracellular amastigotes was quantified by qPCR, and median effective concentration (EC₅₀) was determined using the PROBIT model. The controls included sensitive strains and experimentally derived less sensitive strains generated <i>in vitro</i>, which presented EC₅₀ values up to 7.57-fold higher than the values of the sensitive strains. The isolates were classified into groups according to their <i>in vitro</i> susceptibility profiles using Ward’s hierarchical method. The susceptibility to AmB differed in an intraspecies-specific manner as follows: 28.21% (11/39) of <i>L</i>. <i>(V</i>.<i>) panamensis</i> strains, 50% (3/6) of <i>L</i>. <i>(V</i>.<i>) guyanensis</i> strains and 34.61% (9/26) of <i>L</i>. <i>(V</i>.<i>) braziliensis</i> strains were classified as less sensitive. The latter subset featured three susceptibility groups. We identified Colombian isolates with different AmB susceptibility profiles. In addition, the capacity of species of subgenus <i>Viannia</i> to develop lower susceptibility to AmB was demonstrated <i>in vitro</i>. These new findings should be considered in the pharmacovigilance of AmB in Colombia and South America.</p></div