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

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.

Providing an in vitro depiction of microglial cells challenged with immunostimulatory extracellular vesicles of Naegleria fowleri

“…As described elsewhere, it has been shown that DNA-harboring EVs from malaria parasites could stimulate cytosolic pathogen DNA sensors in host monocytes to elicit a cytokine response ( Sisquella et al, 2017 ). On the other hand, cancer related studies of EV-DNA in plasma and other body fluids for new liquid biopsy applications are a reality ( Malkin and Bratman, 2020 ), and this sets a base for understanding and proposing that DNA from pathogens, included in EV fractions, could also be considered a molecular biomarker as it has been proposed in a study with chronic Chagas disease patients, in which the detection of Trypanosoma cruzi nuclear and kinetoplast DNA was performed in serum circulating EVs ( Lozano et al, 2023 ). For the specific case of primary acute meningoencephalitis, a biomarker present in blood instead of cerebrospinal fluid could make the difference in timely and non-invasive diagnosis, and we suggest this could be studied as it has been found that DNA-carrying EVs could cross the intact blood–brain-barrier, being detectable in peripheral blood ( García-Romero et al, 2017 ).…”