Use of sera cell free DNA (cfDNA) and exovesicle-DNA for the molecular diagnosis of chronic Chagas disease.

Fecha de publicación: 08/09/2023 Fecha Ahead of Print: 08/09/2023

Autores de IIS La Fe

• Eva Calabuig Muñoz

  Autor

• María Dolores Gómez Ruiz

  Autor

• José Miguel Sahuquillo Arce

  Autor

• María Trelis Villanueva

  Autor

• Antonio Osuna Ortega

  Autor

Participantes ajenos a IIS La Fe

• Lozano N
• Samblas MG
• Giménez Martí MJ
• Sequera-Arquelladas S
• Moreno JMM

Grupos

• Infección Grave

  

  Investigador Principal

  María Ángeles Tormo Mas

• Infecciones Respiratorias

  Investigador Principal

  Raúl Méndez Ocaña

• Unidad Mixta de Investigación en Endocrinología, Nutrición y Dietética clínica

  Leader

  Juan Francisco Merino Torres

Abstract

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.

Copyright: © 2023 Lozano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Keywords

• POLYMERASE-CHAIN-REACTION; TRYPANOSOMA-CRUZI INFECTION; CONGENITAL TRANSMISSION; BLOOD-SAMPLES; EXTRACELLULAR VESICLES; SOLID-ORGAN; PCR; EXOSOMES; ANTIGEN; FLUIDS