Sampling is decisive to determination of Leishmania (Viannia) species.

BACKGROUND: Accuracy of molecular tools for the identification of parasites that cause human cutaneous leishmaniasis (CL) could largely depend on the sampling method. Non-invasive or less-invasive sampling methods such as filter paper imprints and cotton swabs are preferred over punch biopsies and lancet scrapings for detection methods of Leishmania based on polymerase chain reaction (PCR) because they are painless, simple, and inexpensive, and of benefit to military and civilian patients to ensure timely treatment. However, different types of samples can generate false negatives and there is a clear need to demonstrate which sample is more proper for molecular assays. METHODOLOGY: Here, we compared the sensitivity of molecular identification of different Leishmania (Viannia) species from Peru, using three types of sampling: punch biopsy, filter paper imprint and lancet scraping. Different composite reference standards and latent class models allowed to evaluate the accuracy of the molecular tools. Additionally, a quantitative PCR assessed variations in the results and parasite load in each type of sample. PRINCIPAL FINDINGS: Different composite reference standards and latent class models determined higher sensitivity when lancet scrapings were used for sampling in the identification and determination of Leishmania (Viannia) species through PCR-based assays. This was consistent for genus identification through kinetoplastid DNA-PCR and for the determination of species using FRET probes-based Nested Real-Time PCR. Lack of species identification in some samples correlated with the low intensity of the PCR electrophoretic band, which reflects the low parasite load in samples. CONCLUSIONS: The type of clinical sample can directly influence the detection and identification of Leishmania (Viannia) species. Here, we demonstrated that lancet scraping samples consistently allowed the identification of more leishmaniasis cases compared to filter paper imprints or biopsies. This procedure is inexpensive, painless, and easy to implement at the point of care and avoids the need for anesthesia, surgery, and hospitalization and therefore could be used in resource limited settings for both military and civilian populations.

Leishmania RNA virus-1 is similarly detected among metastatic and non-metastatic phenotypes in a prospective cohort of American Tegumentary Leishmaniasis.

American Tegumentary Leishmaniasis (ATL) is an endemic and neglected disease of South America. Here, mucosal leishmaniasis (ML) disproportionately affects up to 20% of subjects with current or previous localised cutaneous leishmaniasis (LCL). Preclinical and clinical reports have implicated the Leishmania RNA virus-1 (LRV1) as a possible determinant of progression to ML and other severe manifestations such as extensive cutaneous and mucosal disease and treatment failure and relapse. However, these associations were not consistently found in other observational studies and are exclusively based on cross-sectional designs. In the present study, 56 subjects with confirmed ATL were assessed and followed out for 24-months post-treatment. Lesion biopsy specimens were processed for molecular detection and quantification of Leishmania parasites, species identification, and LRV1 detection. Among individuals presenting LRV1 positive lesions, 40% harboured metastatic phenotypes; comparatively 58.1% of patients with LRV1 negative lesions harboured metastatic phenotypes (p = 0.299). We found treatment failure (p = 0.575) and frequency of severe metastatic phenotypes (p = 0.667) to be similarly independent of the LRV1. Parasite loads did not differ according to the LRV1 status (p = 0.330), nor did Leishmanin skin induration size (p = 0.907) or histopathologic patterns (p = 0.780). This study did not find clinical, parasitological, or immunological evidence supporting the hypothesis that LRV1 is a significant determinant of the pathobiology of ATL.

Chemical study of Adenocalymma axillarum crude leaf extract and isolated compounds

Abstract Adenocalymma axillarum (K.Schum.) L.G. Lohmann is a liana belonging to the family Bignoniaceae. In traditional medicine, the genus Adenocalymma is used to treat fever, skin ailments, and body, joint, and facial muscle pains, and it is also applied as cosmetic. Biological assays conducted with the A. axillarum crude leaf ethanol extract have indicated leishmanicidal activity and absence of cytotoxicity. This study aimed to analyze the A. axillarum leaf ethanol crude extract by high-performance liquid chromatography-high-resolution mass spectrometry- diode array detector (HPLC-HRMS-DAD) and to evaluate the leishmanicidal and cytotoxic activities of this crude extract, its fractions, and isolated compounds. HPLC-HRMS-DAD analysis of this extract revealed that it consisted mainly of flavonoids, with nine major compounds. Extract purification yielded 4-hydroxy-N-methylproline, 6-β-hydroxyipolamiide, quercetin-3-O-robinobioside, hyperin, isorhamnetin-3-O-robinobioside, and 3'-O-methylhyperin, which were identified by Nuclear Magnetic Resonance. The isolated compounds were inactive against Leishmania amazonensis promastigotes and human lung fibroblast cells.

Chalcones identify cTXNPx as a potential antileishmanial drug target.

With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2',6'-dihydroxy-4'-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2',4',6'- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.

Changes in the microbiological diagnosis and epidemiology of cutaneous leishmaniasis in real-time PCR era: A six-year experience in a referral center in Barcelona.

BACKGROUND: Leishmaniasis is a neglected disease caused by different species of the protozoa Leishmania spp. Cutaneous lesions are the most common clinical manifestation. This disease is prevalent in tropical and subtropical areas, including the Mediterranean basin. In Spain, Leishmania (L.) infantum is the only endemic species, but imported cases are often diagnosed. Different classical parasitological methods can be performed for cutaneous leishmaniasis (CL) diagnosis; but currently molecular techniques serve as a relevant tool for the detection and characterization of Leishmania parasites. We aimed to evaluate clinical and epidemiological characteristics of CL diagnosed patients by real-time PCR in a tertiary hospital over a six-year period. METHODOLOGY/PRINCIPAL FINDINGS: Clinical, epidemiological and microbiological data were retrospectively collected and analyzed. In our study, CL was confirmed in 59 (31.4%) out of 188 patients by real-time PCR, showing an increase over recent years: 11 cases of CL between 2014 and 2016 and 48 between 2017 and 2019. Real-time PCR was performed on skin swabs and/or biopsies samples, with a positivity of 38.5% and 26.5%, respectively. Results were 100% concordant when biopsy and skin swab were performed simultaneously. L. (L.) infantum was the most frequent species detected (50%), followed by L. (L.) major (45%) and Viannia subgenus (5%), which were detected only in imported cases. L. (L.) major was almost entirely detected in travelers/migrants from Morocco. Multiple and atypical skin lesions were more common in imported cases than in autochthonous cases (44.4% vs. 21.8%). CONCLUSIONS/SIGNIFICANCE: An increase in both autochthonous and imported CL cases has been observed in past years in our hospital. Molecular techniques assist in improving CL diagnosis and characterization of the Leishmania species, mainly in imported cases.

In Vivo Structure-Function Analysis and Redox Interactomes of Leishmania tarentolae Erv.

Import and oxidative folding of proteins in the mitochondrial intermembrane space differ among eukaryotic lineages. While opisthokonts such as yeast rely on the receptor and oxidoreductase Mia40 in combination with the Mia40:cytochrome c oxidoreductase Erv, kinetoplastid parasites and other Excavata/Discoba lack Mia40 but have a functional Erv homologue. Whether excavate Erv homologues rely on a Mia40 replacement or directly interact with imported protein substrates remains controversial. Here, we used the CRISPR-Cas9 system to generate a set of tagged and untagged homozygous mutants of LTERV from the kinetoplastid model parasite Leishmania tarentolae. Modifications of the shuttle cysteine motif of LtErv were lethal, whereas replacement of clamp residue Cys17 or removal of the kinetoplastida-specific second (KISS) domain had no impact on parasite viability under standard growth conditions. However, removal of the KISS domain rendered parasites sensitive to heat stress and led to the accumulation of homodimeric and mixed LtErv disulfides. We therefore determined and compared the redox interactomes of tagged wild-type LtErv and LtErvΔKISS using stable isotope labeling by amino acids in cell culture (SILAC) and quantitative mass spectrometry. While the Mia40-replacement candidate Mic20 and all but one typical substrate with twin Cx3/9C-motifs were absent in both redox interactomes, we identified a small set of alternative potential interaction partners with putative redox-active cysteine residues. In summary, our study reveals parasite-specific intracellular structure-function relationships and redox interactomes of LtErv with implications for current hypotheses on mitochondrial protein import in nonopisthokonts. IMPORTANCE The discovery of the redox proteins Mia40/CHCHD4 and Erv1/ALR, as well as the elucidation of their relevance for oxidative protein folding in the mitochondrial intermembrane space of yeast and mammals, founded a new research topic in redox biology and mitochondrial protein import. The lack of Mia40/CHCHD4 in protist lineages raises fundamental and controversial questions regarding the conservation and evolution of this essential pathway. Do protist Erv homologues act alone, or do they use the candidate Mic20 or another protein as a Mia40 replacement? Furthermore, we previously showed that Erv homologues in L. tarentolae and the human pathogen L. infantum are not only essential but also differ structurally and mechanistically from yeast and human Erv1/ALR. Here, we analyzed the relevance of such structural differences in vivo and determined the first redox interactomes of a nonopisthokont Erv homologue. Our data challenge recent hypotheses on mitochondrial protein import in nonopisthokonts.

Comparative analysis of the transcriptional responses of five Leishmania species to trivalent antimony.

BACKGROUND: Leishmaniasis is a neglected tropical disease caused by several species of Leishmania. The resistance phenotype of these parasites depends on the characteristics of each species, which contributes to increased therapeutic failures. Understanding the mechanism used by the parasite to survive under treatment pressure in order to identify potential common and specific therapeutic targets is essential for the control of leishmaniasis. The aim of this study was to investigate the expression profiles and potential shared and specific resistance markers of the main Leishmania species of medical importance [subgenus L. (Leishmania): L. donovani, L. infantum and L. amazonensis; subgenus L. (Viannia): L. panamensis and L. braziliensis)] resistant and sensitive to trivalent stibogluconate (SbIII). METHODS: We conducted comparative analysis of the transcriptomic profiles (only coding sequences) of lines with experimentally induced resistance to SbIII from biological replicates of five Leishmania species available in the databases of four articles based on ortholog attribution. Simultaneously, we carried out functional analysis of ontology and reconstruction of metabolic pathways of the resulting differentially expressed genes (DEGs). RESULTS: Resistant lines for each species had differential responses in metabolic processes, compound binding, and membrane components concerning their sensitive counterpart. One hundred and thirty-nine metabolic pathways were found, with the three main pathways comprising cysteine and methionine metabolism, glycolysis, and the ribosome. Differentially expressed orthologous genes assigned to species-specific responses predominated, with 899 self-genes. No differentially expressed genes were found in common among the five species. Two common upregulated orthologous genes were found among four species (L. donovani, L. braziliensis, L. amazonensis, and L. panamensis) related to an RNA-binding protein and the NAD(P)H cytochrome-B5-oxidoreductase complex, associated with transcriptional control and de novo synthesis of linoleic acid, critical mechanisms in resistance to antimonials. CONCLUSION: Herein, we identified potential species-specific genes related to resistance to SbIII. Therefore, we suggest that future studies consider a treatment scheme that is species-specific. Despite the limitations of our study, this is the first approach toward unraveling the pan-genus genetic mechanisms of resistance in leishmaniasis.

Host-Directed Therapies for Cutaneous Leishmaniasis.

Cutaneous leishmaniasis exhibits a wide spectrum of clinical presentations from self-resolving infections to severe chronic disease. Anti-parasitic drugs are often ineffective in the most severe forms of the disease, and in some cases the magnitude of the disease can result from an uncontrolled inflammatory response rather than unrestrained parasite replication. In these patients, host-directed therapies offer a novel approach to improve clinical outcome. Importantly, there are many anti-inflammatory drugs with known safety and efficacy profiles that are currently used for other inflammatory diseases and are readily available to be used for leishmaniasis. However, since leishmaniasis consists of a wide range of clinical entities, mediated by a diverse group of leishmanial species, host-directed therapies will need to be tailored for specific types of leishmaniasis. There is now substantial evidence that host-directed therapies are likely to be beneficial beyond autoimmune diseases and cancer and thus should be an important component in the armamentarium to modulate the severity of cutaneous leishmaniasis.

Synthetic ligustrazine based cyclohexanone and oxime analogs as Anti-Trypanosoma and Anti-Leishmanial agentes

In the present study a series of 34 synthetic ligustrazine-containing α, ß-Unsaturated carbonyl-based compounds and oximes, recognized as anticancer compounds were assessed against protozoa of the Trypanosoma and Leishmania species. Ligustrazine, chemically known as tetramethylpyrazine (TMP), was selected as the core moiety for the synthesis of α, ß-Unsaturated carbonyl-based compounds and these compounds were selected as precursors for the synthesis of new oximes. Some derivates, including 5f and 6i, showed multiple activities against all tested strains. In particular compounds 5f and 8o are the most potent and they are, therefore, potential candidates for trypanosomiasis and leishmaniasis

In vitro inhibitory effect of aluminum phthalocyanine tetrasulfonate chloride against Leishmania (Viannia) Peruviana and Leishmania (Viannia) Braziliensis. / Efecto inhibitorio in vitro de la ftalocianina de aluminio tetrasulfonada clorada frente a Leishmania (Viannia) peruviana y Leishmania (Viannia) braziliensis.

OBJECTIVES: To evaluate the in vitro photodynamic activity of aluminum phthalocyanine tetrasulfonate chloride (AlPcClS4) on promastigotes and amastigotes of Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis. MATERIALS AND METHODS: The activity of photodynamic therapy using AlPcClS4 on Leishmania promastigote and amastigotes was determined by the Methyl Thiazole Tetrazolium (MTT) colorimetric method and quantitative PCR, respectively. RESULTS: Photodynamic treatment showed an inhibitory effect on promastigotes, particularly on Leishmania (V.) peruviana, to a lesser extent on Leishmania (V.) braziliensis and also on intracellular forms of both species. At 24 hours post-radiation, using concentrations of 200 µM and 350 µM, the inhibitory effect on Leishmania (V.) peruviana was 72.9% and 73.9% respectively; at 96 hours the inhibitory effect was of 78.8% and 80.6%, respectively. Regarding intracellular forms, the inhibitory effect on Leishmania (V.) peruviana amastigotes was 57.8% at 72 hours post-treatment, using a concentration of 200 µM. The IC50 was 56.5, 50, 44 and 39.7 µM, at 24, 48, 72 and 96 hours post-radiation, respectively. CONCLUSIONS: Photodynamic therapy using AlPcClS4 against Leishmania species showed encouraging results, mainly on Leishmania (V.) peruviana, suggesting a potential use as an alternative or complement to the usual treatment of tegumentary leishmaniasis. However, new trials are still required to determine the selectivity index for the intracellular form of the parasite, and to develop methods to facilitate the efficient entry of the molecule into the host cell and the parasite.

OBJETIVOS: Evaluar la actividad fotodinámica in vitro de la ftalocianina de aluminio tetrasulfonada clorada (AlPcClS4) sobre promastigotes y amastigotes de Leishmania (Viannia) peruviana y Leishmania (Viannia) braziliensis. MATERIALES Y MÉTODOS: La actividad del tratamiento fotodinámico empleando AlPcClS4 sobre promastigotes y amastigotes de Leishmania fue determinada mediante el método colorimétrico Metil Tiazol Tetrazolium (MTT) y PCR cuantitativo, respectivamente. RESULTADOS: El tratamiento fotodinámico presentó un efecto inhibitorio sobre promastigotes, principalmente sobre Leishmania (V.) peruviana, en menor proporción sobre Leishmania (V.) braziliensis y sobre las formas intracelulares de ambas especies. En Leishmania (V.) peruviana, a las 24 horas posirradiación a 200 µM y 350 µM el efecto inhibitorio fue del 72,9% y 73,9%, respectivamente y a las 96 horas fue del 78,8% y 80,6%, respectivamente. En las formas intracelulares, empleando 200 µM y evaluado a las 72 horas postratamiento, se observó una inhibición del 57,8% de amastigotes de Leishmania (V.) peruviana. El IC50 fue del 56,5; 50; 44; y 39,7 µM, que corresponde a las 24, 48, 72 y 96 horas posirradiación, respectivamente. CONCLUSIONES: El tratamiento fotodinámico empleando AlPcClS4 frente a las especies de Leishmania presentó resultados alentadores principalmente sobre Leishmania (V.) peruviana, lo cual sugiere su potencial uso como alternativa o complemento del tratamiento convencional de la leishmaniasis tegumentaria. Sin embargo, aún se requiere continuar con nuevos ensayos para determinar el índice de selectividad sobre el parásito en su forma intracelular, y desarrollar estrategias que faciliten el ingreso eficiente de la molécula hacia la célula hospedera y al parásito.

Duplex qPCR for Leishmania species identification using lesion imprint on filter paper.

BACKGROUND: American cutaneous leishmaniasis (ACL) is caused by different Leishmania parasites, which stimulate and direct the immune response against the infection. OBJECTIVE: To evaluate the TaqMan probe technology applicability to diagnose and identifying of Leishmania spp. related to the ACL etiology. METHODOLOGY: Through the MEGA 6.0 software, performed an in silico analysis using multiple alignments of Leishmania spp. which were available on GenBank for different genomic targets. The efficiency (e), specificity and detection limit (DL) were calculated for each system, these were associated to compose a duplex-qPCR (DqPCR). The samples of blood, lesion biopsy and lesion imprint on filter paper from patients residing in states of Amazonas (AM) and Pernambuco (PE)-Brazil, (cases and controls) were used to perform the DqPCR technique. The capacity to identify the Leishmania species was determined by comparison with isoenzymes method and sequencing analysis. RESULTS: Internal Transcribed Spacer 1 (rDNA) was the target selected. Two sets of primers and probes were designed and combined: SVS for subgenus Viannia and LaS for L. (L.) amazonensis. The results were: SVSe = 93.24%, SVS DL = 50 fg/µL; LaSe = 89.3%, LaSLD = 5 fg/µL presented 100% of specificity. In total, 236 individuals participated of the present study, wherein were 101 blood samples, 33 biopsies and 147 lesion imprints. The imprint was the most sensitive sample, showing 83.06% of sensitivity, 86.96% of specificity and substantial agreement between the techniques analysis (k = 0.531; p < 0,001). Regarding the species identification, DqPCR and sequencing/isoenzymes have agreed at 100%, since the infection is caused by a single Leishmania species. CONCLUSION: The DqPCR technique was applicable in diagnosis and identification of Leishmania spp. (subgenus Viannia and L. amazonensis). Furthermore, the lesion imprint is less invasive, allowing a fewer discomfort and greater acceptance by the patients, in addition of being low cost and easy handling.

PCR performance for the diagnosis of cutaneous leishmaniasis caused by Leishmania viannia complex using biopsy samples, compared with exudate samples from skin lesions on filter paper.

BACKGROUND: Cutaneous leishmaniasis (CL) is generally diagnosed by molecular methods, including PCR, using biopsy samples, skin scrapings and clinical exudates. In this study, we assessed the PCR performance for diagnosis of CL using skin of biopsy samples vs PCR of skin lesion exudate samples on filter paper and compared the diagnostic concordance of PCR using both sampling methods. METHODS: We assessed the PCR performance using 80 skin biopsy samples and 80 filter paper samples containing exudates from skin lesions obtained from 74 patients with clinical suspicion of CL in Cusco, Peru. RESULTS: : PCR using skin biopsy samples had superior diagnostic accuracy compared with filter paper PCR (62.5% [50/80] vs 38.7% [31/80], respectively; p˂0.005) and the diagnostic concordance between both sampling methods was 'moderate' (kappa coefficient=0.50, 95% CI 0.98 to 1.0). CONCLUSIONS: PCR using biopsy samples remains the standard for diagnosis of CL.

Test accuracy of polymerase chain reaction methods against conventional diagnostic techniques for Cutaneous Leishmaniasis (CL) in patients with clinical or epidemiological suspicion of CL: Systematic review and meta-analysis.

BACKGROUND: Molecular diagnostic tests, notably polymerase chain reaction (PCR), are highly sensitive test for Leishmania detection, which is especially relevant in chronic cutaneous lesion with lower parasite load. An accurate diagnosis is essential because of the high toxicity of the medications for the disease. Nevertheless, diagnosis of cutaneous leishmaniasis (CL) is hampered by the absence of a reference standard. Assuming that the PCR-based molecular tools are the most accurate diagnostic method, the objective of this systematic review was to assess the diagnostic accuracy of PCR-based molecular tools in a meta-analysis of the published literature. METHODOLOGY/PRINCIPAL FINDINGS: A search of the published literature found 142 papers of which only 13 studies met the selection criteria, including conventional PCR, real-time PCR, Loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), polymorphism-specific PCR (PS-PCR). The sensitivities of the individual studies ranged from 61% to 100%, and specificities ranged from 11% to 100%. The pooled sensitivities of PCR in smears were 0.95 (95% CI, 0.90 to 0.98), and the specificity was 0.91(95% CI, 0.70 to 0.98). In general population, estimates were lower in aspirates, skin biopsies and swab samples with 0.90 (95% CI, 0.80 to 0.95) and 0.87 (95% CI, 0.76 to 0.94) for sensitivity and specificity, respectively. The specificity was lower in consecutive studies, at 0.88 (95% CI, 0.59 to 0.98) and its CI were wider. CONCLUSIONS/SIGNIFICANCE: No statistically significant differences between the accuracy in smears, aspirate, skin biopsies or swabs samples were observed. Therefore, a simple smear sample run by PCR, instead more invasive samples, may be enough to obtain a positive diagnosis of CL. The results for PCR in all samples type confirm previous reports that consider PCR as the most accurate method for the diagnosis of CL.

DNA-based detection of Leishmania and Crithidia species isolated from humans in cutaneous and post-kala-azar dermal leishmaniasis from Shiraz and Kharameh, southern Iran.

BACKGROUND & OBJECTIVES: Leishmania major and L. tropica are the main pathogens of cutaneous leishmaniasis (CL) in several rural and some urban regions of Iran, respectively. The aim of this study was to detect Leishmania species, and update the distribution data of these species in humans suspected to CL in two endemic foci in southern Iran. METHODS: From March 2016 to March 2017, 276 positive samples from of 350 suspected cases were diagnosed and compared by different diagnostic methods, viz. microscopy, culture, and PCR. In PCR assay, four different gene identifications were performed including minicircle kDNA, and cysteine protease B genes for Leishmania detection, and glyceraldehyde-3-phosphate dehydrogenase, and internal transcribed spacer 1 genes for Crithidia detection. RESULTS: In total, 68% (235/350) and 65.3% (177/271) of patients suspected of leishmaniasis were positive by microscopy and cultivation methods. In PCR assay, L. major, and L. tropica were detected in 86.2% (238/276), and 13.1% (36/276) of CL cases, respectively. Also, dermal L. infantum strain was isolated from 0.7% (2/276) of post-kala-azar dermal leishmaniasis patients. In addition, Crithidia fasciculata was detected in two CL patients chronically infected with L. major. INTERPRETATION & CONCLUSION: It appears that the epidemiology of CL has changed during the last decades and can complicate the control strategy aspects of CL in southern Iran. Therefore, more epidemiological, ecological, and gene polymorphism studies are needed to understand the pathogenic role of these species in human, as a main host of leishmaniasis in Iran.

Increasing putative vector importance of Trichophoromyia phlebotomines (Diptera: Psychodidae)

Despite some phlebotomines being well recognised as vectors of leishmaniasis agents, vector importance of those belonging to the genus Trichophoromyia has not been extensively studied. The present study provides evidence regarding the putative vector role played by some species of Trichophoromyia on leishmanine enzootics, based on literature reports and findings obtained from field experiments conducted in the ecotopes of Pará State, Brazil. The species Th. ubiquitalis, Th. velascoi, Th. auraensis, Th. ininii and Th. brachipyga possess minimal criteria to be included in the list of suspected leishmanine vectors. However, knowledge on man-biting behavior, substantiation of vector competence and determination of epidemiological implications are limited for all of the above mentioned species. Published studies together with present data draw attention to prioritize these phlebotomine species in entomological surveillance programs and studies on experimental susceptibility to Leishmania spp. infection.

Clinical aspects and diagnosis of leishmaniasis in equids: a systematic review and meta-analysis / Aspectos clínicos e diagnóstico da leishmaniose em equídeos: uma revisão sistemática e meta-análise

Abstract Leishmaniases are a group of diseases of zoonotic importance caused by over 20 species of protozoa of the genus Leishmania, in which domestic dogs are considered to be the main reservoir for the disease. However, the involvement of other vertebrates as reservoirs for these parasites has also been investigated. Therefore, the objective of the present study was to carry out a systematic review with meta-analysis on occurrences of leishmaniasis in equids. The case reports described animals with cutaneous symptoms of leishmaniasis (papules, nodules, ulcers or crusts) that regressed spontaneously, located mainly on the head and limbs, from which three species of protozoa were identified in the lesions: Leishmania braziliensis, Leishmania infantum and Leishmania siamensis. In turn, the meta-analysis showed a combined prevalence of 25%, although with high heterogeneity among the studies, which was attributed to the use of different methods for diagnosing the disease. Leishmaniasis in equids is a benign disease but it should be included in the differential diagnosis of cutaneous diseases among these species. Seroepidemiological studies are important in investigating and monitoring suspected exposure of these hosts to the parasite, especially in endemic areas. However, there is also a need to standardize diagnostic methods.

Resumo As leishmanioses são um grupo de doenças de importância zoonótica causadas por mais de 20 espécies de protozoários do gênero Leishmania, sendo o cão doméstico considerado o principal reservatório da doença. No entanto, diversas pesquisas têm investigado o envolvimento de outros vertebrados como reservatórios do parasita. Portanto, o objetivo do presente estudo foi realizar uma revisão sistemática com meta-análise da ocorrência de leishmaniose em equídeos. Os relatos de caso descreviam animais com sintomas cutâneos de leishmaniose (pápulas, nódulos, úlceras, crostas) que regrediam espontaneamente, localizadas principalmente na cabeça e membros, sendo identificadas três espécies do protozoário nas lesões: Leishmania braziliensis, Leishmania infantum e Leishmania siamensis. Por sua vez, a meta-análise evidenciou uma prevalência combinada de 25%, porém com alta heterogeneidade entre os estudos, atribuída às diferenças nos métodos utilizados no diagnóstico da doença. A leishmaniose em equídeos é uma doença benigna, porém deve ser incluída no diagnóstico diferencial de doenças cutâneas nessas espécies. Os estudos soroepidemiológicos são importantes para investigar e monitorar a suspeita de exposição desses hospedeiros ao parasita, principalmente em áreas endêmicas, porém há necessidade de padronização dos métodos de diagnóstico.

Case Report: Mucocutaneous Leishmaniasis Masquerading as Idiopathic Midline Granulomatous Disease.

Mucocutaneous leishmaniasis (MCL) is a rare infection caused by several species within the genus Leishmania. We present a patient with multifocal MCL masquerading as idiopathic midline granulomatous disease, featuring the unusual complication of ocular leishmaniasis, as a result of prolonged immunosuppressive therapy. We review clinical features, diagnosis, and treatment of this syndrome.

Surveillance of phlebotomine fauna and Didelphis marsupialis (Didelphimorphia: Didelphidae) infection in an area highly endemic for visceral leishmaniasis in Colombia / Vigilancia de la fauna flebotomínea e infección de Didelphis marsupialis (Didelphimorphia: Didelphidae) en un área de alta endemia de leishmaniasis visceral en Colombia

Abstract Introduction: The study of the interaction between the parasite, the vector and the mammalian hosts, including man, allows to understand the behavior of the leishmaniases. Objective: To determine the presence of Lutzomyia species and to detect the Leishmania infection in Didelphis marsupialis in an endemic area for visceral leishmaniasis. Materials and methods: Phlebotomine fauna and individuals of D. marsupialis were collected with CDC and Tomahawk™ traps, respectively. The species of Lutzomyia were identified using the Young and Duncan key (1994). Ear and tail biopsies and blood samples from D. marsupialis were taken to identify the Leishmania species by amplifying a fragment of the gene associated with the 70 kD heat shock protein. Results: Seven Lutzomyia species were identified: Lu. evansi, Lu. gomezi, Lu. panamensis, Lu. dubitans, Lu. cayennensis cayennensis, Lu. rangeliana and Lu. trinidadensis. The first three species have epidemiological importance in Colombia because of their implications in the transmission of the Leishmania parasite. Sixty-five tissue samples from 19 D. marsupialis individuals were negative for Leishmania spp. Conclusions: The presence of the Lutzomyia species that have been identified as vectors for Leishmania inside and around houses in the village of El Bledo, in El Carmen de Bolívar represents a risk of infection. Furthermore, the presence of Lu. panamensis is reported for first time in El Carmen de Bolívar in Colombia. Although the lack of detection of Leishmania spp. in D. marsupialis samples may suggest that D. marsupialis does not play an important role in the transmission cycle of Leishmania in this region, it is necessary to carry out further longitudinal studies to confirm this hypothesis.

Resumen Introducción. El estudio de la interacción entre el parásito, el vector y los huéspedes mamíferos, incluido el hombre, permite entender el comportamiento de la leishmaniasis. Objetivo. Determinar la presencia de especies del género Lutzomyia y detectar la infección por Leishmania spp. en Didelphis marsupialis en un área endémica de leishmaniasis visceral. Materiales y métodos. Se recolectaron flebotomíneos y D. marsupialis con trampas CDC y Tomahawk™, respectivamente. Las especies de Lutzomyia se identificaron usando la clave de Young y Duncan, 1994. Se tomaron biopsias de oreja, cola y muestras de sangre de D. marsupialis para diagnosticar Leishmania spp. mediante la amplificación de un fragmento del gen de la proteína de choque térmico de 70 kD. Resultados. Se identificaron siete especies de Lutzomyia: Lu. evansi, Lu. gomezi, Lu. panamensis, Lu. dubitans, Lu. cayennensis cayennensis, Lu. rangeliana y Lu. trinidadensis. Las tres primeras especies son reconocidas como vectores en el país por estar implicadas en la transmisión de Leishmania spp. En total, 65 muestras de tejidos de oreja, cola y de sangre provenientes de 19 D. marsupialis fueron negativas para Leishmania spp. en la PCR-HSP70. Conclusiones. La presencia de flebotomíneos con importancia epidemiológica en la zona evaluada representa un riesgo de transmisión. Asimismo, Lu. panamensis es reportada por primera vez en El Bledo (Carmen de Bolívar). La ausencia de Leishmania spp. en D. marsupialis podría sugerir que esta especie no tiene un papel importante en el ciclo de transmisión de Leishmania en la vereda El Bledo, por lo que es necesario profundizar en estudios longitudinales para corroborar esta hipótesis.

Geographical distribution of Leishmania species in Colombia, 1985-2017 / Distribución geográfica de las especies de Leishmania en Colombia, 1985-2017

Abstract Introduction: Knowledge of the geographical distribution of Leishmania species allows guiding the sampling to little-studied areas and implementing strategies to define risk zones and priority areas for control. Objective: Given that there is no publication that collects this information, the search, review, and compilation of the available scientific literature that has identified species in Colombia is presented in this paper. Materials and methods: A bibliographic search was performed in PubMed, Web of Knowledge, Google Scholar, SciELO and LILACS with the terms "(Leishmania OR Leishmaniasis) AND species AND Colombia", without restrictions on publication year, language or infected organism; records of national scientific events and repositories of theses from Colombian universities were also included. Results: Eighty-six scientific documents published between 1985 and 2017 were found in which the species of Leishmania and their geographical origin were indicated. The species reported, in descending order of frequency, were: Leishmania (Viannia) panamensis, L. (V.) braziliensis, L. (V.) guyanensis, L. (Leishmania) infantum, L. (L.) amazonensis, L. (L.) mexicana, L. (V.) colombiensis, L. (V.) lainsoni and L. (V.) equatorensis; the last three were found with the same frequency. Leishmania species were reported from 29 departments. Conclusion: Information on the distribution of Leishmania species in Colombia is limited; therefore, it is necessary to gather existing data and propose studies that consolidate the distribution maps of Leishmania species in Colombia. This would allow the detection of areas where species have not been identified as well as the comparison of existing parasite and vector distributions.

Resumen Introducción. El conocimiento de la distribución geográfica de las especies de Leishmania permite orientar el muestreo hacia áreas poco estudiadas e implementar estrategias para detectar zonas de riesgo y áreas prioritarias de control. Objetivo. Dado que no existe una publicación que reúna esta información, se planteó la revisión y compilación de la literatura científica disponible de estudios de identificación de especies del país. Materiales y métodos. Se llevó a cabo una búsqueda bibliográfica en PubMed, Web of Knowledge, Google Académico, SciELO y Lilacs con los términos "(Leishmania OR Leishmaniasis) AND especie AND Colombia", así como en memorias de eventos científicos nacionales y repositorios de tesis y trabajos de grado de universidades del país. Resultados. Se encontraron 86 documentos científicos publicados entre 1985 y 2017, en los cuales se informaron la especie de Leishmania y el origen geográfico. Las especies circulantes reportadas, en su orden de frecuencia, fueron: Leishmania (Viannia) panamensis, L. (V.) braziliensis, L. (V.) guyanensis, L. (Leishmania) infantum, L. (L.) amazonensis, L. (L.) mexicana, L. (V.) colombiensis, L. (V.) lainsoni y L. (V.) equatorensis, las últimas tres, con igual frecuencia. Los reportes proceden de 29 departamentos. Conclusión. La información de la distribución de las especies de Leishmania en Colombia es limitada. Por lo tanto, se necesita reunir los datos existentes y plantear trabajos que permitan consolidar el mapa de distribución de las especies en el país, lo cual permitiría detectar las zonas sin información de las especies circulantes y establecer la concordancia entre su distribución y la de los vectores.

Synthesis and Biological Studies of (+)-Liquiditerpenoic Acid A (Abietopinoic Acid) and Representative Analogues: SAR Studies.

The first semisynthesis and biological profiling of the new abietane diterpenoid (+)-liquiditerpenoic acid A (abietopinoic acid) (7) along with several analogues are reported. The compounds were obtained from readily available methyl dehydroabietate (8), which was derived from (-)-abietic acid (1). Biological comparison was conducted according to the different functional groups, leading to some basic structure-activity relationships (SAR). In particular, the ferruginol and sugiol analogues 7 and 10-16 were characterized by the presence of an acetylated phenolic moiety, an oxidized C-7 as a carbonyl, and a different functional group at C-18 (methoxycarbonyl, carboxylic acid, and hydroxymethyl). The biological properties of these compounds were investigated against a panel of six representative human tumor solid cells (A549, HBL-100, HeLa, SW1573, T-47D, and WiDr), five leukemia cellular models (NALM-06, KOPN-8, SUP-B15, UoCB1, and BCR-ABL), and four Leishmania species ( L. infantum, L. donovani, L. amazonensis, and L. guyanensis). A molecular docking study pointed out some targets in these Leishmania species. In addition, the ability of the compounds to modulate GABAA receptors (α1ß2γ2s) is also reported. The combined findings indicate that these abietane diterpenoids offer a source of novel bioactive molecules with promising pharmacological properties from cheap chiral-pool building blocks.