Use of cfDNA and exovesicle-DNA for the molecular diagnosis of chronic Chagas disease

Chagas disease is, among others, considered a neglected tropical disease. Given the magnitude of the human movements that have occurred in recent years from Central and South America to other countries, Chagas should now be considered a disease of worldwide distribution, in which the transmission of the parasite is restricted to transplacental transmission or blood or organ donations from infected people. Parasite detection in chronically ill patients is restricted to serological tests that only determine previous contact and not the presence of the parasite, especially in those patients undergoing treatment evaluation or in newborns. In this study, we evaluate the use of nucleic acids from both circulating serum exovesicles and cell-free DNA (cfDNA) from 448 serum samples from immunologically diagnosed chronic chagasic patients, which were re-evaluated by nested PCR on the amplicons resulting from amplification with kDNA-specific primers 121F-122R. Of the total number of samples selected, 50 were used to isolate and purify exovesicles from circulating serum and cell-free DNA (cfDNA). When the nucleic acids thus purified were assayed as a template and amplified with primers 121F-122R and SAT, a percentage positivity of 100% was obtained for all positive samples assayed with the kDNA-specific primers and 96% when SAT primers were used. However, isolation of cfDNA for T. cruzi and amplification with SAT primers also showed 100% positivity. Hence, both samples can be used in those cases where it is necessary to demonstrate the active presence of the parasite. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial University of Granada 201102400000408 and 672/CEIH/2018 ### Funding Statement This research was funded by the ERANET program, Research in prevention of congenital Chagas disease parasitological, placental and immunological markers ERANET17 HLH 0142. Instituto Carlos III, Ministerio de Sanidad, Gobierno de Spain. 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. Ministerio de Ciencia y Tecnologia of the government of Spain funded the project PGC2018 099424 BI00 and The financial support given by the project ABIO 350 UGR18 Proyect Programa Operativo FEDER de Andalucia JJAA 2014 2020 ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All of the patients gave written informed consent before starting the study and all protocols were approved on March 4, 2011, the study was expanded to include the use to isolate and purify exovesicles from circulating serum and cell-free DNA (cfDNA) on September 26, 2018, with registration numbers 201102400000408 and 672/CEIH/2018 respectively, by the ethical committee of Granada University. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present work are contained in the manuscript in supplementary material