Use of sera cell free DNA (cfDNA) and exovesicle-DNA for the molecular diagnosis of chronic Chagas disease Lozano Rodríguez, Noelia Gomez-Samblas, Mercedes Osuna Carrillo De Albornoz, Antonio This research was funded by the ERANet program, Research in prevention of congenital Chagas disease: parasitological, placental and immunological markers (ERANet17/HLH-0142 (Cochaco). Instituto Carlos III, Ministerio de Sanidad, Gobierno de Espana. Fundacion Ramon Areces "Interactoma de las exovesiculas de T. cruzi y de los inmunocomplejos que forman con las celulas del hospedador: implicaciones en la patologia de la enfermedad de Chagas (2019)". PreChag y el titulo Exovesiculas circulantes como marcadoras de diagnostico, PREcoz de la Enfermedad de CHAGas del XXI Concurso Nacional para la adjudicacion de Ayudas a la Investigacion en Ciencias de la Vida y de la Materia (2022). Ministerio de Ciencia y Tecnologia of the government of Spain funded the project PGC2018-099424-B-I00 and The financial support given by the proyect A-BIO-350-UGR18 I+D+i Proyect "Programa Operativo FEDER de Andalucia JJAA" 2014-2020. Chagas disease, a neglected tropical disease, is now considered a worldwide health concern as a result of migratory movements from Central and South America to other regions that were considered free of the disease, and where the epidemiological risk is limited to transplacental transmission or blood or organ donations from infected persons. Parasite detection in chronically ill patients is restricted to serological tests that only determine infection by previous infection and not the presence of the parasite, especially in patients undergoing treatment evaluation or in newborns. We have evaluated the use of nucleic acids from both circulating exovesicles and cell-free DNA (cfDNA) from 50 samples twice randomly selected from a total of 448 serum samples from immunologically diagnosed patients in whom the presence of the parasite was confirmed by nested PCR on amplicons resulting from amplification with kinetoplastid DNA-specific primers 121F-122R. Six samples were randomly selected to quantify the limit of detection by qPCR in serum exovesicles. When the nucleic acids thus purified were assayed as a template and amplified with kinetoplastid DNA and nuclear satellite DNA primers, a 100% positivity rate was obtained for all positive samples assayed with kDNA-specific primers and 96% when SAT primers were used. However, isolation of cfDNA for Trypanosoma cruzi and amplification with SAT also showed 100% positivity. The results demonstrate that serum exovesicles contain DNA of mitochondrial and nuclear origin, which can be considered a mixed population of exovesicles of parasitic origin. The results obtained with serum samples prove that both cfDNA and Exovesicle DNA can be used to confirm parasitaemia in chronically ill patients or in samples where it is necessary to demonstrate the active presence of the parasite. The results confirm for the first time the existence of exovesicles of mitochondrial origin of the parasite in the serum of those affected by Chagas disease. 2023-12-13T13:22:22Z 2023-12-13T13:22:22Z 2023-09-08 journal article Lozano N, Samblas MG, Calabuig E, Giménez Martí MJ, Gómez Ruiz MD, Arce JMS, et al. (2023). Use of sera cell free DNA (cfDNA) and exovesicle-DNA for the molecular diagnosis of chronic Chagas disease. PLoS ONE 18(9): e0282814. [https://doi.org/10.1371/journal.pone.0282814] https://hdl.handle.net/10481/86162 10.1371/journal.pone.0282814 eng http://creativecommons.org/licenses/by/4.0/ open access Atribución 4.0 Internacional PLOS