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

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.

[In vitro study of the antimicrobial properties of a silver ion-releasing polyurethane foam]. / Estudio in vitro de las propiedades antimicrobianas de una espuma de poliuretano que libera iones de plata.

INTRODUCTION: The antimicrobial properties of a silver ion (Ag+)-releasing polyurethane foam were evaluated using different microorganisms. The diffusion of Ag+ from the medium, as well as any possible cytotoxicity on human cells, was also studied. MATERIAL AND METHODS: Silver release from V.A.C. GranuFoam Silver(®) was assessed by using inductively coupled plasma mass spectrometry (ICP-MS). An in vitro experimental study was designed to evaluate the bactericide capacity using lethal dose curves on A. baumannii, P. aeruginosa, S. maltophilia, K. pneumoniae, E. coli, P. mirabilis, methicillin resistant S. aureus, E. faecium, S. pyogenes and C. minutissimum. A cytotoxicity study was also performed on human fibroblasts. RESULTS: The silver release showed an exponential curve with a stable meseta phase after 3 hours, with levels of 0.22-0.24 mg/l. A reduction of 99.9% of all the gram-negatives was achieved at 3 hours. The reduction was greater than 99% at 2 hours in S. pyogenes and C. minutissimum, at 6h in S. aureus and at 14 h in E. faecium. In an in vivo simulation model, these reductions were achieved in 6 hours in the gram negatives and 24h in the gram positives. The silver concentrations were no cytotoxic to human fibroblasts, with no differences being observed between the cells exposed to Ag+ and the controls (p=.7) CONCLUSION: V.A.C. Granufoam Silver(®) releases bactericide concentrations of Ag+ that did not damage human fibroblasts. It appears to be a good alternative for the control and prevention of local infections.