Introducción: La enfermedad de Chagas, causada por Trypanosoma cruzi, afecta entre 6 y 8 millones de personas en todo el mundo. Los tratamientos actuales plantean problemas de seguridad. La variabilidad del tamizaje fenotípico in vitro en cuanto a cepas del parásito, estadios del ciclo vital y condiciones experimentales dificultan la comparación de resultados e impiden el desarrollo de nuevos tratamientos. Objetivo: Identificar condiciones experimentales críticas en el modelo de epimastigotes de T. cruzi y proponer criterios para armonizar los procedimientos experimentales. Metodología: Análisis de 63 artículos de investigación (106 ensayos) de revistas indexadas (2008-2023), sobre actividad antiparasitaria frente a epimastigotes. Resultados: Los estudios utilizan diversas formas evolutivas, principalmente amastigotes y epimastigotes. A pesar de avances en las técnicas de cultivo, establecer condiciones uniformes para todos los estadios sigue siendo inviable. Proponemos focalizar formas parasitarias específicas. Conclusiones: Para armonizar condiciones experimentales, proponemos optimizar: cepas, medio de cultivo (y enriquecimiento), parámetros de incubación (temperatura y frecuencia de sustitución del medio). Del mismo modo, las condiciones de prueba farmacológica, como concentración del inóculo infectante, especificaciones de placa de cultivo, tiempo de exposición al tratamiento y método de cuantificación del parásito, facilitan comparación de resultados y aceleran el desarrollo de nuevos tratamientos.

Acute respiratory infections and other infectious diseases causing acute febrile syndrome are major public health concerns in Colombia, particularly among vulnerable populations such as the Wayuu Indigenous community in Manaure, La Guajira. To investigate their viral etiology, 55 nasopharyngeal swabs and 58 serum samples were collected from febrile Wayuu individuals in Manaure. RT-qPCR screening identified Coronavirus, Enteroviruses, Adenovirus, and Influenza A/B in respiratory samples, while no arboviruses were detected in serum. Sixteen representative samples underwent metatranscriptomic next-generation sequencing (mtNGS) using the Chan-Zuckerberg ID (CZ-ID) platform. This analysis confirmed RT-qPCR findings and additionally revealed six viral contigs related to Orthopicobirnavirus hominis. Sequencing coverage enabled the reconstruction of a consensus RdRp segment, which was phylogenetically compared with sequences from diverse hosts. The virus clustered within genogroup 1, alongside Colombian isolates linked to severe acute respiratory infection. The absence of strict host-specific clustering suggests possible interspecies transmission. These findings underscore the complementary roles of targeted and unbiased approaches: RT-qPCR detected common respiratory viruses, whereas mtNGS uncovered a virus previously unreported in this community. Overall, mtNGS emerges as a powerful tool to support viral surveillance and provide baseline evidence in indigenous populations, emphasizing the need to decentralize advanced molecular diagnostics and strengthen public health capacity in Colombia.

Acute diarrheal disease (ADD) caused by parasites and Tuberculosis (TB) remain major public health concerns in vulnerable indigenous communities with limited access to sanitation, safe water, and healthcare, and where humans, animals and the environment interact closely. Using a One Health framework, this study investigated TB and Intestinal parasites in human, animal and environmental samples from 15 Wayuu indigenous communities in Manaure, La Guajira. A total of 190 samples, including human sputum and feces, animal milk and feces, soil and drinking water, were analyzed according to sample type, preservation suitability, and availability using parasitological concentration techniques, qPCR for helminth detection, metatranscriptomic sequencing, Xpert ^® MTB/RIF assay, and mycobacterial culture. Mycobacterium tuberculosis was detected in 8.3% of human sputum samples, with no evidence of rifampicin resistance, whereas Mycobacterium bovis was not detected in animal milk. Human fecal samples analyzed by microscopy showed Blastocystis sp. and the Entamoeba histolytica/Entamoeba dispar complex (38.8% each), followed by Giardia (19.4%), Hymenolepis nana and Trichuris trichiura (5.1% each) and Hymenolepis diminuta (1%). Commensal parasites were also identified, with Entamoeba coli (46.9%) being the most frequent species, indicating inadequate sanitary conditions and poor hygiene practices. Co-infections were common in humans (60.2%). In animal fecal samples, strongylids (66.7%), amoebas (16.7%) and Giardia (8.3%) were observed. Giardia sp. was detected in 2.38% of soil samples by microscopy, supporting environmental circulation, whereas no parasites were detected in water sediments. Multiplex qPCR detected Trichuris trichiura DNA in human feces and Trichuris spp. DNA in soil and sheep fecal samples. Metatranscriptomic analysis of 22 human fecal samples revealed a high diversity and frequency of parasitic protozoa (90.9%), with Blastocystis spp. being the most frequent (81.8%). Additionally, reads of free-living amoebae, including Acanthamoeba spp. (10%) and Naegleria spp. (5%) were detected in community drinking water sources. These findings suggest active transmission of TB and parasitic-associated ADD in Wayuu communities and highlight the need for integrated surveillance and culturally appropriate interventions focused on sanitation, hygiene, veterinary services and community health education to improve the living and health conditions of these vulnerable populations.

Chagas disease, caused by Trypanosoma cruzi, poses a significant global health burden. Current treatment relies solely on benznidazole and nifurtimox, both with limited efficacy and significant adverse effects. In vitro phenotype-based screening enables identification of alternative treatments. While epimastigote-based screening is not suitable for primary drug discovery, it provides valuable complementary data for compound evaluation. However, the lack of standardized experimental conditions across studies compromises reproducibility and inter-laboratory comparability. Here, we optimized experimental conditions for T. cruzi epimastigote drug susceptibility assays. Trypanosoma cruzi epimastigotes (strain Y) were cultured under systematically varied conditions to optimize medium composition, serum supplementation, medium replacement frequency, inoculum density, and treatment exposure time. Epimastigote growth peaked on day 8, declining thereafter. Low initial parasite inoculum showed reduced growth and high variability, while high parasite densities formed aggregates. Growth rates were similar across media, but Roswell Park Memorial Institute medium could promote trypomastigote-like forms. Fetal bovine serum (FBS) enhanced growth rates, with a positive correlation between serum concentration and proliferation. Three-day medium replacement maintained >98% epimastigotes. Benznidazole exhibited an IC50 of 16.61 ± 0.38 µM under optimized conditions. Optimal conditions include: Liver Infusion Tryptose or Brain Heart Infusion medium with 10% FBS, medium replacement every 3 days, 1 × 106 parasites/ml inoculum, and 72-h treatment at 28°C. The assay demonstrated robustness across microplate designs, producing consistent results with both U-bottom and flat-bottom 96-well plates. Under these optimized conditions, benznidazole serves as an effective positive control.

Acute diarrheal disease (ADD) caused by parasites and TB represent a significant public health burden worldwide and in Colombia, particularly affecting indigenous populations who are at high risk of contracting these diseases due to the social, environmental, and cultural conditions in which they live. Materials: Fifteen Wayuu indigenous communities in four areas of Manaure, in La Guajira, were subject to intervention; with prior informed consent, environmental samples and samples from individuals with clinical symptoms were collected. A total of 156 samples of human and animal feces, soil, and sediment from drinking water were analyzed for microscopic using the Kato–Katz and formalin–ether concentration techniques, 109 samples were analyzed by qPCR for the detection of helminths and 23 for metatranscriptomics targeting protozoan parasites and helminths. Additionally, 36 sputum samples from patients with respiratory symptoms were tested using Xpert/MTB Rif, and 37 milk samples were tested for M. bovis. Results: Among the samples tested for tuberculosis, a positivity rate of 8.3% was found, in all cases with sensitivity to rifampicin; M. bovis was not found in animal milk. Microscopic analysis of human samples revealed pathogenic parasites, the most common being Blastocystis spp. and the Entamoeba hystolitica/Entamoeba dispar complexeach with 38.8% (n=38), Giardia spp. with 19.4%, Hymenolepis nanaand Trichuris trichiura each with 5.1%. Commensal parasites were also identified as indicator of poor sanitary conditions. Co-infection with intestinal parasites was common in humans at 60.2%. In microscopic analysis of animals fecal samples, revealed a high incidence of Uncinaria spp. with 58.3%, amoebas 16.7% and Giardia 8.3%; this latter is also found in soil. Metatranscriptomics showed a high frequency of intestinal parasites in fecal samples (90.9%), with Blastocystis spp. being the most frequent (81.8%) with notable intra-species diversity, followed by Entamoeba histolytica (54.5%) and Giardia duodenalis (31.8%), and detected free-living amoebae in community water sources, highlighting potential health risks associated with exposure to untreated water in low-sanitation settings. Conclusions: The Wayuu communities studied show a significant burden of tuberculosis and intestinal parasitic infections, likely associated with poor sanitary conditions and environmental factors that facilitate their transmission. Although TB prevalence was moderate, with no evidence of rifampicin resistance or the circulation of M. bovis in milk, the high prevalence of intestinal parasites, including co-infections and their detection in humans, animals, and the environment suggest active transmission in the region. These findings highlight the need to implement comprehensive interventions in water, sanitation, and hygiene, along with surveillance and health education strategies with an intercultural approach, aimed at improving the conditions of these vulnerable populations.