The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR–Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23-and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null muta...

Introducción: La Escherichia coli enterotoxigénica (ETEC) causa diarrea en niños menores de 5 años. Su diagnóstico se basa en la detección de las toxinas LT o ST. Actualmente, las técnicas moleculares son usadas, pero su aplicación en campo es difícil. La amplificación isotérmica con recombinasa y polimerasa (RPA) es una técnica reciente que se usa en la detección de varios patógenos. La RPA tiene potencial para su uso en campo porque puede funcionar entre 37° y 42°C, la amplificación es rápida y los reactivos pueden ser almacenados sin refrigeración por varias semanas. Objetivo: Desarrollar un método para el diagnóstico de Escherichia coli enterotoxigénica (ETEC) en muestras de heces mediante la detección simultánea de los genes para las toxinas LT y ST usando la amplificación con recombinasa y polimerasa (RPA). Métodos: Se diseñaron y seleccionaron cebadores ...

Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing...

Low- and middle-income countries (LMICs) are significantly affected by SARS-CoV-2, partially due to their limited capacity for local production and implementation of molecular testing. Here, we provide detailed methods and validation of a molecular toolkit that can be readily produced and deployed using laboratory equipment available in LMICs. Our results show that lab-scale production of enzymes and nucleic acids can supply over 50,000 tests per production batch. The optimized one-step RT-PCR coupled to CRISPR-Cas12a-mediated detection showed a limit of detection of 102 ge/μL in a turnaround time of 2 h. The clinical validation indicated an overall sensitivity of 80%–88%, while for middle and high viral load samples (Cq ≤ 31) the sensitivity was 92%–100%. The specificity was 96%–100% regardless of viral load. Furthermore, we show that the toolkit can be used with the mobile lab...

En nuestro país, en la última década, se han detectado 8000 casos por año de leishmaniasis cutánea (LC), siendo Leishmania braziliensis considerada una de las especies más importantes, debido a su prevalencia, asociación a falla terapéutica, y riesgo de generar leishmaniasis mucosa.Actualmente no existe una vacuna efectiva contra LC para uso en humanos, a pesar de los esfuerzos a nivel mundial. Estudios en vacunas basadas en epítopes facilitaron el desarrollo de herramientas inmuno-informáticas para predecir epítopes potencialmente inmunogénicos. Pocos estudios han utilizado métodos inmunoinformáticos con el fin de identificar epítopes de células T, a partir del proteoma de Leishmania, además de su validación experimental. La mayoría de estudios se enfocaron en especies de Leishmania del Viejo Mundo, mientras que pocos en especies del Nuevo Mundo, particularmente L. br...

Viruses are the most abundant biological entities on Earth and play a significant role in the evolution of many organisms and ecosystems. In pathogenic protozoa, the presence of viruses has been linked to an increased risk of treatment failure and severe clinical outcome. Here, we studied the molecular epidemiology of the zoonotic disease cutaneous leishmaniasis in Peru and Bolivia through a joint evolutionary analysis of Leishmania braziliensis and their dsRNA Leishmania virus 1. We show that parasite populations circulate in tropical rainforests and are associated with single viral lineages that appear in low prevalence. In contrast, groups of hybrid parasites are geographically and ecologically more dispersed and associated with an increased prevalence, diversity and spread of viruses. Our results suggest that parasite gene flow and hybridization increased the frequency of parasite-vi...