2056. Trypanosama cruzi DNA Detection by PCR in Dried Blood Spots Preserved in Filter Paper

Abstract

BackgroundEarly diagnosis of Congenital Chagas Disease is important to initiate efficient treatment, so it is necessary to develop techniques with lower detection limits and greater specificity like PCR. The lack of validated protocols and the need to send samples to be analyzed in experienced centers makes dried blood spots preserved in filter paper an attractive alternative for the conservation and handling of samples. The aim of this study was to optimize the detection of Trypanosoma cruzi DNA from dried blood spots preserved in filter paper.MethodsFixed sections of Whatman filter paper with different concentrations of T. cruzi were prepared (104/mL at 10–1/mL) and stored at room temperature, 4 and −20°C in the presence or absence of a desiccant. Samples (8 mm2) were taken at 7, 60, 90 and 240 days of preservation. Endpoint PCR, targeting 18S gene, was used for the detection of T. cruzi DNA directly on the filter paper.Results T. cruzi DNA was detected at all sampling times up to the 102/mL concentration independently of conservation. The effect of humidity was observed at 240 days preservation with the observation of faded bands in agarose gels. For the 101/mL concentration, T. cruzi DNA was detected only at 7 days regardless of preservation. When comparing T. cruzi DNA detection using increasing sections of filter paper (8, 16 and 24 mm2), T. cruzi DNA was detected in all areas tested in the concentration of 101 parasites/mL and only when using 24 mm2 for the concentration of 1 parasite/mL.ConclusionDried blood spots preserved in filter paper allowed detection of T. cruzi DNA by endpoint PCR in the different conservation conditions up to 8 months. The detection of parasite DNA was improved by increasing the area of filter paper tested. The conservation of blood on filter paper would provide a safe transport of samples at room temperature to distant specialized laboratories to perform diagnosis using molecular techniques.Disclosures All authors: No reported disclosures.