-Adapting a molecular isothermal amplification reaction to develop a simplified Trypanosoma cruzi detection method

LAROCCA LUCIANA; WERBAJH SANTIAGO; CARRILLO C; STOLOWICZ FABIANA; RUIZ D; FRACCAROLI L; VOJNOV ADRIAN

Buenos Aires

Congreso; XXIX Reunión de la Sociedad Argentina de Protozoología, en el marco de la Reunión Conjunta de Sociedades de Biociencias.; 2018

Reunión Conjunta de Sociedades de Biociencias

Chagas disease, originally from Latin America, is caused by Trypanosomacruzi. It is estimated that 15000 babies are born withcongenital Chagas around the world each year. Early detectionand treatment of congenital Chagas disease increases the therapy´s effectiveness. However, serological methods of diagnosis areeffective only after 9 months of age, when maternal antibodies havebeen completely removed. On the other hand, molecular diagnosticstrategies, applicable for newborns, require infrastructure, skilledpersonnel and expensive equipment, factors that restrict their useto few health centers.The objective of this work was to develop a molecular amplificationtechnique to detect T. cruzi DNA with high sensitivity and specificitythat would be simple and stable enough to be used on point of care(?POC?) conditions.The first step consisted in selecting target sequences and primerdesign, using specific bioinformatics software. Then, the in vitroreaction was set up testing: a- the reaction conditions; b- sensitivitywith different types of samples; and c- specificity, using differentstrains of T. cruzi, other phylogenetically related parasites, humancells and cells unrelated to the parasite or host. Finally, the reactionwas adapted to make it simpler and suitable for all health careconditions (different equipment, infrastructure, etc.). The read-outmethods were simplified, showing a changing color on a lateral flowdipstick (LFD) system.Two of the 6 sets of designed primers, from repetitive DNA fragments,showed high specificity (with no cross reactions) and highsensitivity (~1fg of template), along different types of templates(DNA, parasites, artificially inoculated samples, etc.) and using analyticalread-out (gel electrophoresis), color changes and LFD.These results encourage us to continue developing a test for Chagasdisease as well as for other infectious diseases whose currentdiagnosis methods also need improvement and simplification.Keywords: Chagas; Trypanosoma Cruzi; Point Of Care Conditions;Molecular Isothermal Amplification.