Unveiling the Genetic Blueprint of a Desert Scorpion: A Chromosome-level Genome of Hadrurus arizonensis Provides the First Reference for Parvorder Iurida.

Over 400 million years old, scorpions represent an ancient group of arachnids and one of the first animals to adapt to life on land. Presently, the lack of available genomes within scorpions hinders research on their evolution. This study leverages ultralong nanopore sequencing and Pore-C to generate the first chromosome-level assembly and annotation for the desert hairy scorpion, Hadrurus arizonensis. The assembled genome is 2.23 Gb in size with an N50 of 280 Mb. Pore-C scaffolding reoriented 99.6% of bases into nine chromosomes and BUSCO identified 998 (98.6%) complete arthropod single copy orthologs. Repetitive elements represent 54.69% of the assembled bases, including 872,874 (29.39%) LINE elements. A total of 18,996 protein-coding genes and 75,256 transcripts were predicted, and extracted protein sequences yielded a BUSCO score of 97.2%. This is the first genome assembled and annotated within the family Hadruridae, representing a crucial resource for closing gaps in genomic knowledge of scorpions, resolving arachnid phylogeny, and advancing studies in comparative and functional genomics.

Mutational Profile and Retinal Phenotypes of PCARE-Related Cone-Rod Dystrophies in a Mexican Cohort.

Purpose: The aim of the study is to describe the genotype and phenotype of a Mexican cohort with PCARE-related retinal disease. Methods: The study included 14 patients from 11 unrelated pedigrees with retinal dystrophies who were demonstrated to carry biallelic pathogenic variants in PCARE. Visual assessment methods included best corrected visual acuity, color fundus photography, Goldmann visual field test, kinetic perimetry, dark/light adapted chromatic perimetry, full-field electroretinography, autofluorescence imaging, and spectral domain-optical coherence tomography imaging. Genetic screening was performed either by gene panel sequencing or by exome sequencing. Results: According to the results of multimodal imaging and functional tests, all 14 patients were diagnosed with cone-rod dystrophy. Six different PCARE pathogenic alleles were identified in our cohort, including three novel mutations: c.3048_3049del (p.Tyr1016∗), c.3314_3315del (p.Ser1105∗), and c.551A > G (p.His184Arg). Notably, alleles p.His184Arg, p.Arg613∗, and p.Arg984∗ were present in 18 of the 22 (82%) PCARE alleles from probands in our cohort. Conclusion: Our work expands the PCARE mutational profile by identifying three novel pathogenic variants causing retinal dystrophy. While phenotypic variations occurred among patients, a cone-rod dystrophy pattern was observed in all affected individuals.

Post-Anesthesia Cognitive Dysfunction in Mice Is Associated with an Age-Related Increase in Neuronal Intracellular [Ca2+]-Neuroprotective Effect of Reducing Intracellular [Ca2+]: In Vivo and In Vitro Studies.

Background: Postoperative cognitive dysfunction (POCD) is a common disorder after general anesthesia in elderly patients, the precise mechanisms of which remain unclear. Methods: We investigated the effect of isoflurane with or without dantrolene pretreatment on intracellular calcium concentration ([Ca2+]i), reactive oxygen species (ROS) production, cellular lactate dehydrogenase (LDH) leak, calpain activity, and cognitive function using the Morris water maze test of young (3 months), middle-aged (12-13 months), and aged (24-25 months) C57BL6/J mice. Results: Aged cortical and hippocampal neurons showed chronically elevated [Ca2+]i compared to young neurons. Furthermore, aged hippocampal neurons exhibited higher ROS production, increased LDH leak, and elevated calpain activity. Exposure to isoflurane exacerbated these markers in aged neurons, contributing to increased cognitive deficits in aged mice. Dantrolene pretreatment reduced [Ca2+]i for all age groups and prevented or significantly mitigated the effects of isoflurane on [Ca2+]i, ROS production, LDH leak, and calpain activity in aged neurons. Dantrolene also normalized or improved age-associated cognitive deficits and mitigated the cognitive deficits caused by isoflurane. Conclusions: These findings suggest that isoflurane-induced cytotoxicity and cognitive decline in aging are linked to disruptions in neuronal intracellular processes, highlighting the reduction of [Ca2+]i as a potential therapeutic intervention.

Promiscuous CYP87A enzyme activity initiates cardenolide biosynthesis in plants.

Cardenolides are specialized, steroidal metabolites produced in a wide array of plant families1,2. Cardenolides play protective roles in plants, but these molecules, including digoxin from foxglove (Digitalis spp.), are better known for treatment of congenital heart failure, atrial arrhythmia, various cancers and other chronic diseases3-9. However, it is still unknown how plants synthesize 'high-value', complex cardenolide structures from, presumably, a sterol precursor. Here we identify two cytochrome P450, family 87, subfamily A (CYP87A) enzymes that act on both cholesterol and phytosterols (campesterol and ß-sitosterol) to form pregnenolone, the first committed step in cardenolide biosynthesis in the two phylogenetically distant plants Digitalis purpurea and Calotropis procera. Arabidopsis plants overexpressing these CYP87A enzymes ectopically accumulated pregnenolone, whereas silencing of CYP87A in D. purpurea leaves by RNA interference resulted in substantial reduction of pregnenolone and cardenolides. Our work uncovers the key entry point to the cardenolide pathway, and expands the toolbox for sustainable production of high-value plant steroids via synthetic biology.

Neglected no longer: Phylogenomic resolution of higher-level relationships in Solifugae.

Advanced sequencing technologies have expedited resolution of higher-level arthropod relationships. Yet, dark branches persist, principally among groups occurring in cryptic habitats. Among chelicerates, Solifugae ("camel spiders") is the last order lacking a higher-level phylogeny and have thus been historically characterized as "neglected [arachnid] cousins". Though renowned for aggression, remarkable running speed, and xeric adaptation, inferring solifuge relationships has been hindered by inaccessibility of diagnostic morphological characters, whereas molecular investigations have been limited to one of 12 recognized families. Our phylogenomic dataset via capture of ultraconserved elements sampling all extant families recovered a well-resolved phylogeny, with two distinct groups of New World taxa nested within a broader Paleotropical radiation. Divergence times using fossil calibrations inferred that Solifugae radiated by the Permian, and most families diverged prior to the Paleogene-Cretaceous extinction, likely driven by continental breakup. We establish Boreosolifugae new suborder uniting five Laurasian families, and Australosolifugae new suborder uniting seven Gondwanan families using morphological and biogeographic signal.

Old and cold: Diverse phylogenomic datasets support an ancient transantarctic dispersive route on the scorpion family Bothriuridae in temperate Gondwana.

In this contribution we try to unveil the diversification process of Bothriuridae in temperate Gondwana through dated phylogenomic analyses using UCE and transcriptomics, and including in the analyses species of genera Urophonius and Cercophonius, the most closely related genera of Bothriuridae from South America and Australia respectively. Additionally we explored the hypothesis that the winter activity period of some species of Urophonius, as well as the cold environmental preferences of this genus, could be related to the climatic conditions of the time frame and area in which it evolved. Genus Urophonius was recovered as sister group to Cercophonius using amino acids and UCE. The time frame obtained for the split between South American and Australian bothriurids is 94 Ma., which suggests a dispersal event through temperate Gondwana, before the final breakup of the land bridge of South America-Antarctica-Australia ca. 35 Ma. The split between summer and winter species of Urophonius, taking place at 64 Ma, is considered representative to the turnover time from the summer activity period to the winter activity period in some species of the genus. This time frame is compatible with a period of global warming of the late Cretaceous greenhouse episode that could have triggered this change.

Congruence between ultraconserved element-based matrices and phylotranscriptomic datasets in the scorpion Tree of Life.

Scorpions are ancient and historically renowned for their potent venom. Traditionally, the systematics of this group of arthropods was supported by morphological characters, until recent phylogenomic analyses (using RNAseq data) revealed most of the higher-level taxa to be non-monophyletic. While these phylogenomic hypotheses are stable for almost all lineages, some nodes have been hard to resolve due to minimal taxonomic sampling (e.g. family Chactidae). In the same line, it has been shown that some nodes in the Arachnid Tree of Life show disagreement between hypotheses generated using transcritptomes and other genomic sources such as the ultraconserved elements (UCEs). Here, we compared the phylogenetic signal of transcriptomes vs. UCEs by retrieving UCEs from new and previously published scorpion transcriptomes and genomes, and reconstructed phylogenies using both datasets independently. We reexamined the monophyly and phylogenetic placement of Chactidae, sampling an additional chactid species using both datasets. Our results showed that both sets of genome-scale datasets recovered highly similar topologies, with Chactidae rendered paraphyletic owing to the placement of Nullibrotheas allenii. As a first step toward redressing the systematics of Chactidae, we establish the family Anuroctonidae (new family) to accommodate the genus Anuroctonus.

Reproductive performance of hair ewes and rams implanted with melatonin previous to the anestrus season in northwest Mexico.

Ovine reproductive behavior depends on annual photoperiodic cycle and its impact on endogenous melatonin secretion. In this regard, exogenous melatonin administration previous to the physiological anestrus period could modify the reproductive performance of sheep in the northwest of Mexico. Two independent studies were performed to evaluate such hypothesis in hair sheep implanted with melatonin prior to the anestrus season in latitudes 24° and 25° of Mexico. Study 1 involved 15 rams assigned to one of three treatments receiving 0mg (n=5), 18mg (n=5), or 36mg (n=5) of melatonin subcutaneously. Study variables were measured monthly since implantation (d0) and included testosterone concentration, scrotal circumference, mass motility, individual motility, and sperm concentration. Study 2 included 50 ewes assigned to one of two treatments receiving 0mg (n=25) or 18mg (n=25) of melatonin subcutaneously. In ewes, progesterone concentration and the frequency of females in anestrus were measured during the implantation (-30d), as well as at the beginning (0d) and at the end (45d) of the mating period, while pregnancy rate was determined by ultrasonography 45d after. Continuous variables were analyzed using a mixed effects model considering treatment, time, and the treatment by time interaction as fixed effects. Animal nested within treatment was the random effect. Binary variables were analyzed using the chi-square test. In males, melatonin improved testosterone and sperm concentrations (P<0.05), while in females, a 28% higher pregnancy rate was observed in implanted ewes (P<0.05). Therefore, melatonin enhanced reproductive parameters in both sexes and its exogenous administration previous to the anestrus season in northwest Mexico could be more effective in rams.

Mother-to-infant microbiota transmission and infant microbiota development across multiple body sites.

Early-life microbiota seeding and subsequent development is crucial to future health. Cesarean-section (CS) birth, as opposed to vaginal delivery, affects early mother-to-infant transmission of microbes. Here, we assess mother-to-infant microbiota seeding and early-life microbiota development across six maternal and four infant niches over the first 30 days of life in 120 mother-infant pairs. Across all infants, we estimate that on average 58.5% of the infant microbiota composition can be attributed to any of the maternal source communities. All maternal source communities seed multiple infant niches. We identify shared and niche-specific host/environmental factors shaping the infant microbiota. In CS-born infants, we report reduced seeding of infant fecal microbiota by maternal fecal microbes, whereas colonization with breastmilk microbiota is increased when compared with vaginally born infants. Therefore, our data suggest auxiliary routes of mother-to-infant microbial seeding, which may compensate for one another, ensuring that essential microbes/microbial functions are transferred irrespective of disrupted transmission routes.

Biocatalytic routes to stereo-divergent iridoids.

Thousands of natural products are derived from the fused cyclopentane-pyran molecular scaffold nepetalactol. These natural products are used in an enormous range of applications that span the agricultural and medical industries. For example, nepetalactone, the oxidized derivative of nepetalactol, is known for its cat attractant properties as well as potential as an insect repellent. Most of these naturally occurring nepetalactol-derived compounds arise from only two out of the eight possible stereoisomers, 7S-cis-trans and 7R-cis-cis nepetalactols. Here we use a combination of naturally occurring and engineered enzymes to produce seven of the eight possible nepetalactol or nepetalactone stereoisomers. These enzymes open the possibilities for biocatalytic production of a broader range of iridoids, providing a versatile system for the diversification of this important natural product scaffold.

Biosynthesis of strychnine.

Strychnine is a natural product that, through isolation, structural elucidation and synthetic efforts, shaped the field of organic chemistry. Currently, strychnine is used as a pesticide to control rodents1 because of its potent neurotoxicity2,3. The polycyclic architecture of strychnine has inspired chemists to develop new synthetic transformations and strategies to access this molecular scaffold4, yet it is still unknown how plants create this complex structure. Here we report the biosynthetic pathway of strychnine, along with the related molecules brucine and diaboline. Moreover, we successfully recapitulate strychnine, brucine and diaboline biosynthesis in Nicotiana benthamiana from an upstream intermediate, thus demonstrating that this complex, pharmacologically active class of compounds can now be harnessed through metabolic engineering approaches.

Bacterial and fungal communities in tracheal aspirates of intubated COVID-19 patients: a pilot study.

Co-infections with bacterial or fungal pathogens could be associated with severity and outcome of disease in COVID-19 patients. We, therefore, used a 16S and ITS-based sequencing approach to assess the biomass and composition of the bacterial and fungal communities in endotracheal aspirates of intubated COVID-19 patients. Our method combines information on bacterial and fungal biomass with community profiling, anticipating the likelihood of a co-infection is higher with (1) a high bacterial and/or fungal biomass combined with (2) predominance of potentially pathogenic microorganisms. We tested our methods on 42 samples from 30 patients. We observed a clear association between microbial outgrowth (high biomass) and predominance of individual microbial species. Outgrowth of pathogens was in line with the selective pressure of antibiotics received by the patient. We conclude that our approach may help to monitor the presence and predominance of pathogens and therefore the likelihood of co-infections in ventilated patients, which ultimately, may help to guide treatment.

Chronic Elevation of Skeletal Muscle [Ca2+]i Impairs Glucose Uptake. An in Vivo and in Vitro Study.

Skeletal muscle is the primary site of insulin-mediated glucose uptake through the body and, therefore, an essential contributor to glucose homeostasis maintenance. We have recently provided evidence that chronic elevated intracellular Ca2+ concentration at rest [(Ca2+)i] compromises glucose homeostasis in malignant hyperthermia muscle cells. To further investigate how chronic elevated muscle [Ca2+]i modifies insulin-mediated glucose homeostasis, we measured [Ca2+]i and glucose uptake in vivo and in vitro in intact polarized muscle cells from glucose-intolerant RYR1-p.R163C and db/db mice. Glucose-intolerant RYR1-p.R163C and db/db mice have significantly elevated muscle [Ca2+]i and reduced muscle glucose uptake compared to WT muscle cells. Dantrolene treatment (1.5 mg/kg IP injection for 2 weeks) caused a significant reduction in fasting blood glucose levels and muscle [Ca2+]i and increased muscle glucose uptake compared to untreated RYR1-p.R163C and db/db mice. Furthermore, RYR1-p.R163C and db/db mice had abnormal basal insulin levels and response to glucose-stimulated insulin secretion. In vitro experiments conducted on single muscle fibers, dantrolene improved insulin-mediated glucose uptake in RYR1-p.R163C and db/db muscle fibers without affecting WT muscle fibers. In muscle cells with chronic elevated [Ca2+]i, GLUT4 expression was significantly lower, and the subcellular fraction (plasma membrane/cytoplasmic) was abnormal compared to WT. The results of this study suggest that i) Chronic elevated muscle [Ca2+]i decreases insulin-stimulated glucose uptake and consequently causes hyperglycemia; ii) Reduced muscle [Ca2+]i by dantrolene improves muscle glucose uptake and subsequent hyperglycemia; iii) The mechanism by which chronic high levels of [Ca2+]i interfere with insulin action appears to involve the expression of GLUT4 and its subcellular fractionation.

Phylogenomics of Scorpions Reveal Contemporaneous Diversification of Scorpion Mammalian Predators and Mammal-Active Sodium Channel Toxins.

Scorpions constitute a charismatic lineage of arthropods and comprise more than 2500 described species. Found throughout various tropical and temperate habitats, these predatory arachnids have a long evolutionary history, with a fossil record that began in the Silurian. While all scorpions are venomous, the asymmetrically diverse family Buthidae harbors nearly half the diversity of extant scorpions, and all but one of the 58 species that are medically significant to humans. However, the lack of a densely sampled scorpion phylogeny has hindered broader inferences of the diversification dynamics of scorpion toxins. To redress this gap, we assembled a phylogenomic data set of 100 scorpion venom gland transcriptomes and genomes, emphasizing the sampling of highly toxic buthid genera. To infer divergence times of venom gene families, we applied a phylogenomic node dating approach for the species tree in tandem with phylostratigraphic bracketing to estimate the minimum ages of mammal-specific toxins. Our analyses establish a robustly supported phylogeny of scorpions, particularly with regard to relationships between medically significant taxa. Analysis of venom gene families shows that mammal-active sodium channel toxins (NaTx) have independently evolved in five lineages within Buthidae. Temporal windows of mammal-targeting toxin origins are correlated with the basal diversification of major scorpion mammal predators such as shrews, bats, and rodents. These results suggest an evolutionary model of relatively recent diversification of buthid NaTx homologs in response to the diversification of scorpion predators. [Adaptation; arachnids; phylogenomic dating; phylostratigraphy; venom.].

Phylogeny-Aware Chemoinformatic Analysis of Chemical Diversity in Lamiaceae Enables Iridoid Pathway Assembly and Discovery of Aucubin Synthase.

Countless reports describe the isolation and structural characterization of natural products, yet this information remains disconnected and underutilized. Using a cheminformatics approach, we leverage the reported observations of iridoid glucosides with the known phylogeny of a large iridoid producing plant family (Lamiaceae) to generate a set of biosynthetic pathways that best explain the extant iridoid chemical diversity. We developed a pathway reconstruction algorithm that connects iridoid reports via reactions and prunes this solution space by considering phylogenetic relationships between genera. We formulate a model that emulates the evolution of iridoid glucosides to create a synthetic data set, used to select the parameters that would best reconstruct the pathways, and apply them to the iridoid data set to generate pathway hypotheses. These computationally generated pathways were then used as the basis by which to select and screen biosynthetic enzyme candidates. Our model was successfully applied to discover a cytochrome P450 enzyme from Callicarpa americana that catalyzes the oxidation of bartsioside to aucubin, predicted by our model despite neither molecule having been observed in the genus. We also demonstrate aucubin synthase activity in orthologues of Vitex agnus-castus, and the outgroup Paulownia tomentosa, further strengthening the hypothesis, enabled by our model, that the reaction was present in the ancestral biosynthetic pathway. This is the first systematic hypothesis on the epi-iridoid glucosides biosynthesis in 25 years and sets the stage for streamlined work on the iridoid pathway. This work highlights how curation and computational analysis of widely available structural data can facilitate hypothesis-based gene discovery.

Comprehensive Species Sampling and Sophisticated Algorithmic Approaches Refute the Monophyly of Arachnida.

Deciphering the evolutionary relationships of Chelicerata (arachnids, horseshoe crabs, and allied taxa) has proven notoriously difficult, due to their ancient rapid radiation and the incidence of elevated evolutionary rates in several lineages. Although conflicting hypotheses prevail in morphological and molecular data sets alike, the monophyly of Arachnida is nearly universally accepted, despite historical lack of support in molecular data sets. Some phylotranscriptomic analyses have recovered arachnid monophyly, but these did not sample all living orders, whereas analyses including all orders have failed to recover Arachnida. To understand this conflict, we assembled a data set of 506 high-quality genomes and transcriptomes, sampling all living orders of Chelicerata with high occupancy and rigorous approaches to orthology inference. Our analyses consistently recovered the nested placement of horseshoe crabs within a paraphyletic Arachnida. This result was insensitive to variation in evolutionary rates of genes, complexity of the substitution models, and alternative algorithmic approaches to species tree inference. Investigation of sources of systematic bias showed that genes and sites that recover arachnid monophyly are enriched in noise and exhibit low information content. To test the impact of morphological data, we generated a 514-taxon morphological data matrix of extant and fossil Chelicerata, analyzed in tandem with the molecular matrix. Combined analyses recovered the clade Merostomata (the marine orders Xiphosura, Eurypterida, and Chasmataspidida), but merostomates appeared nested within Arachnida. Our results suggest that morphological convergence resulting from adaptations to life in terrestrial habitats has driven the historical perception of arachnid monophyly, paralleling the history of numerous other invertebrate terrestrial groups.

Exploring the potential relationship between collagen cross-linking and impaired myocardial relaxation in Marfan syndrome: An observational study using serum biomarkers.

BACKGROUND: Increased collagen cross-linking (CCL) has been described in hypertensive cardiomyopathy by means of reduced serum ratio of serum carboxyterminal telopeptide of collagen type I (CITP) to matrix metalloproteinase-1 (MMP1). Previous studies have demonstrated the existence of primary impaired diastole in patients with Marfan syndrome (MFS), but little is known about the pathophysiology of this condition. METHODS: 60 MFS patients (without previous cardiovascular surgery or significant valvular regurgitation) and 24 healthy controls (age and sex-matched) were enrolled. All participants underwent a comprehensive transthoracic echocardiographic study, including left atrial and left ventricular speckle-tracking strain analysis. CITP and MMP1 were measured in peripheral blood. RESULTS: All participants had normal diastolic function according to guidelines. Peak left atrial strain in the reservoir phase (LASr) was significantly reduced in the MFS cohort compared to controls (32.2 ± 9.4 vs 43.9 ± 7.0%; p < 0.001). Serum CITP and CITP:MMP1 ratio were lower among MFS patients, showing significant correlations with LASr (R = 0.311; p = 0.020 and R = 0.437; p = 0.001, respectively). The MFS cohort was divided into quartiles of LASr. MFS patients in the lowest quartile of LASr (<26%) had significantly lower values of CITP:MMP1 ratio compared to the other quartiles. CONCLUSIONS: The analysis of serum biomarkers revealed the presence of increased CCL in association with reduced LASr in the MFS cohort. Our results suggest that excessive CCL may play a role in the development of primary myocardial impairment in these patients. Future studies are needed to confirm our findings and evaluate the prognostic role of CCL markers in the MFS population.

Identificación de especies de Leishmania mediante PCR en tiempo real acoplada a curvas de fusión de alta resolución

RESUMEN Introducción: La leishmaniasis es una enfermedad causada por parásitos del género Leishmania. En Colombia se han informado 10 especies patógenas. El diagnóstico parasitológico tradicional basado en la observación de los parásitos no permite identificar la especie, por lo cual se deben emplear métodos moleculares, entre ellos la reacción en cadena de la polimerasa o PCR convencional, pero esta presenta algunas limitaciones y requiere extensos periodos de tiempo para la obtención de resultados, que en ocasiones no son concluyentes. Objetivo: Evaluar un método basado en PCR en tiempo real acoplado a curva de temperatura de desnaturalización media de alta resolución (PCR-HRM) que permita el diagnóstico y la identificación simultánea de parásitos del género Leishmania en muestras clínicas de humanos y en cultivos in vitro de manera sensible y específica. Métodos: Se estandarizó una PCR-HRM, mediante la cual se evaluaron 237 muestras clínicas, 98 clasificadas como positivas y 139 como negativas, parasitológicamente por directo y/o cultivo. Las tipificaciones fueron comparadas con los resultados en paralelo obtenidos de una variante de la PCR, realizando cortes al amplicon que generó un fragmento de restricción de longitud polimórfica o PCR-RFLP que había sido previamente estandarizada. Resultados: Se logró implementar una PCR-HRM para el diagnóstico e identificación de especies de Leishmania, logrando un 100 % de concordancia con las tipificaciones obtenidas por PCR-RFLP. Incluso, se logró detectar e identificar el parásito en muestras diagnosticadas como negativas por los métodos convencionales. Se encontró que con un porcentaje de confiabilidad superior al 95 %, se lograron tipificar 91 muestras de 98; de estas el 81,63 % de los casos fueron L. panamensis, el 11,22% L. braziliensis e indeterminadas el 7,14 % de los casos. Conclusiones: La PCR-HRM es un buen método que permite la identificación de las especies más prevalentes en Colombia, comparando temperaturas medias de desnaturalización específicas según la especie de Leishmania involucrada.

ABSTRACT Introduction: Leishmaniasis is a disease caused by parasites of the genus Leishmania. Ten pathogenic species have been reported in Colombia. Traditional parasite diagnosis based on observation of the parasites does not make it possible to identify the species. Therefore, it is necessary to use molecular methods, among them conventional polymerase chain reaction or PCR, but this test presents some limitations and requires long periods of time to obtain results which sometimes are not conclusive. Objective: Evaluate a method based on real time PCR coupled with high resolution mean denaturalization temperature curve analysis (HRM-PCR) for the diagnosis and simultaneous identification of parasites of the genus Leishmania in clinical samples from humans and in vitro cultures in a sensitive and specific manner. Methods: Standardization was performed of an HRM-PCR with which 237 clinical samples were evaluated, 98 classified as positive and 139 as negative, by direct parasitological examination and/or culture. The typing obtained was compared with parallel results from a PCR variant, making cuts on the amplicon that generated a restriction fragment length polymorphism or PCR-RFLP previously standardized. Results: An HRM-PCR could be implemented for the diagnosis and identification of Leishmania species, achieving 100% concordance with the typing obtained by PCR-RFLP. It was even possible to detect and identify the parasite in samples diagnosed as negative by conventional methods. Of the total 98 samples, 91 could be typed with a percentage of reliability above 95%. Of these, 81.63% of the cases were L. panamensis, 11.22% were L. braziliensis and 7.14 % were indeterminate. Conclusions: HRM-PCR is a good method to identify the species most prevalent in Colombia, comparing specific mean denaturalization temperatures according to the Leishmania species involved.