Alternative methodology for nucleic acid extraction from filter paper dried blood spots (DBS) for application over Bio-Manguinho's Malaria Diagnostic Kit

Introduction:Malaria is an infectious, febrile, and acute disease caused by Plasmodium protozoa.In addition to transmission by Anopheles mosquitoes, transmission can also occur through blood transfusion.Bio-Manguinhos is in the final stage of developing a Malaria Typing Kit for molecular diagnosis by PCR for the 3 main Plasmodium causing human malaria and one of the sample types to be included in this test is from dried blood spot (DBS) samples on filter paper. Objectives:The objective of this work is to develop a DNA extraction protocol from dried blood samples extracted from filter paper and test it on the Malaria Typing Molecular Kit, under development by Bio-Manguinhos.Methodology: A total of 730 dried blood samples were received from 20 health units from Colombia.For diagnosis, a methodology was developed that consists of pre-processing the samples with the inclusion of a washing step of the sample in the paper three times with Tris-EDTA pH 8.0 (TE), followed by the addition of 20 L of Proteinase K in 200 L of Lysis solution (both from the NATPLUS Kit).The samples were incubated for 25 minutes at 60C, being mixed every 5 minutes, and subsequently maintained at 95C for 5 minutes for proteinase K inactivation.After pre-processing, the eluate was extracted by the NATPLUS Platform, tested using the Malaria Typing Molecular Kit and analyzed using the Design & Analysis (DA) software from Thermofisher. Results:The new methodology obtained a mean Ct of 25.22 versus 26.19 from the commercial one for the endogenous control (RNAse P -RP).Of the 730 samples from Colombia evaluated, 729 (99.86%) detected RP with a mean Ct of 25.62 and range between 22.17 and 29.45.A total of 23 (3.15%) malariapositive samples were detected, being 17 (2.33%) for P. malariae, 3 (0.41%) for P. falciparum, 3 (0.41%)only for General Malaria, and 1 (0.13%) for co-infection of P. malariae and P. falciparum.These Positive samples will be re-tested using an secondary methodology. Conclusion:The protocol developed by Lamol proved to be similar sensitive in detecting RP and enabled the detection of 23 positive samples in DBS for malaria, from Colombia.The inclusion of another sample type has the advantage of being a minimally invasive method, easy to collect and transport from remote regions.