Standardization of DNA extraction from paraffinized spleen samples: molecular diagnosis of human malaria

• Published in: Malaria journal Vol. 22; no. 1; pp. 1 - 361
• Main Authors: Pacheco Souza, Raimunda Sandra, dos Reis, Monique F, de Lima Ferreira, Luiz Carlos, Morais, Manuela C, Lima, Antonio Kassio S, Albuquerque Barbosa, Laila Rowena, de Melo, Gisely Cardoso, de Lacerda, Marcus Vinicius Guimaraes
• Format: Journal Article
• Language: English
• Published: London BioMed Central Ltd 27-11-2023; BioMed Central; BMC
• Subjects: Alcohol; Analysis; Asymptomatic; Care and treatment; Deoxyribonucleic acid; Diagnosis; DNA; DNA sequencing; Ethanol; Genes; Histology; Human diseases; Malaria; Medical diagnosis; Nucleotide sequence; Nucleotide sequencing; Parasites; Pathology; Patients; Plasmodium; Plasmodium vivax; Polymerase chain reaction; Preservation; Risk factors; Spleen; Splenectomy; Standardization; Testing; Vector-borne diseases; Brazil
• ISSN: 1475-2875; 1475-2875
• DOI: 10.1186/s12936-023-04764-3

Background Plasmodium vivax is the main species responsible for human malaria in Brazil, and one of its manifestations is splenic malaria, though there are still challenges in its diagnosis. The present study aimed to standardize Plasmodium sp. DNA extraction from histological slices of spleen and diagnosis using real-time qPCR. Methods This study performed a microtomy of a paraffin-embedded spleen as a positive control for P. vivax from a patient who had been previously diagnosed with the parasite. The sample was deparaffinized with xylol and ethanol, then DNA extraction was performed with two commercial kits. qPCR was carried out with the Taqman system for detection of Plasmodium sp. and was made species-specific using PvmtCOX1 gene. From 2015 to 2019, 200 spleen samples were obtained from trauma patients subjected to splenectomy in Manaus, Amazonas. All the samples were tested for cell-free human DNA (cfDNA). Results The deparaffinization and the Plasmodium vivax DNA extraction method was successfully standardized, and the control sample was positive for P. vivax. Of the 200 samples, all qPCRs were negative, but they were positive for human PCR. Conclusion Paraffinization is practical and efficient for the preservation of samples, but the formation of bonds between proteins and DNA makes extraction difficult. Despite this, in this study, it was possible to standardize a method of DNA extraction for detecting P. vivax.