Standardization of Trypanosoma cruzi DNA extraction and purification protocol from samples collected on Whatman 903 filter paper to Chagas disease diagnosis

Chagas disease (CD) caused by the parasite Trypanosoma cruzi , belongs to the so-called neglected diseases group. In Argentina about 1,500 children are born with congenital Chagas disease per year. The diagnosis of CD in the newborn relies on the ability to detect parasites in the blood by microscopic observation, as the serological tests are ruled out because of the presence of maternal antibodies. CD treatment is more effective during the acute phase of infection. Early diagnosis and treatment of the disease is thus very important. The Argentinian National Program for early detection of metabolic diseases uses Whatman903 filter paper for blood sampling. This type of sample collection presents many advantages as the use of low blood volumes, minimal biological risk, and easy storage and transportation. The objective of the study was to evaluate the conservation efficiency of blood samples on filter paper in order to access good sensitivity on qPCR results for the detection of T. cruzi . To standardize the procedure, negative samples of blood were infected artificially with serial dilutions of trypomastigotes forms of T. cruzi from the TcVI strain obtained by cell culture in Vero cells. Concentrations between 50000 and 5 parasites/mL were prepared and loaded in filter paper for analysis. DNA extraction was conducted by the QIAamp DNA Mini Kit from QIAGEN. For qPCR, a method based on TaqMan technology was used, with a multiplex reaction for quantification of T. cruzi satellite DNA and an internal amplification control (IAC). The detection limit found from our results was 400 parasites/mL, demonstrating that this method could be a reliable option for the diagnosis of congenital CD by the detection of T. cruzi in blood collected in filter paper. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was funded by financial support of the Hospital de Ninos Ricardo Gutierrez ### 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: The Ethics Committee and Review Board of the Hospital de Ninos Ricardo Gutierrez approved this study All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. 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 relevant data are within the manuscript and its Supporting Information files.