Development of a multiplex molecular and serological panel for the diagnosis of clinically important pathogens for xenotransplantation

• Grant number: 25/04517-5
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: June 01, 2025
• End date: May 31, 2028
• Field of knowledge: Biological Sciences - Genetics - Animal Genetics
• Principal Investigator: Silvano Mario Attilio Raia
• Grantee: Luiz Gustavo Cano Munhoz
• Host Institution: Faculdade de Medicina (FM). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 21/11872-5 - Science for Development Center in Xenotransplantation, AP.CCD

Abstract

The scarcity of organ donations worldwide is considered a major barrier to transplants. In view of this, xenotransplantation has emerged as an important alternative to overcome the shortage of organs needed for transplants. Pigs have rapid maturation, numerous offspring and physiological characteristics similar to humans, which make them ideal candidates to be xenotransplant donors. However, these animals can transmit zoonotic pathogens capable of causing serious damage to the organ recipient. Therefore, it is necessary to constantly monitor the animals, the environment ("pig facilities") and the individuals who manage them, in order to maintain the status: SPF (Specific pathogen-free) and DPF (Designated pathogen-free).Currently, real-time polymerase chain reaction (qPCR) is considered the best alternative for detecting viral pathogens, due to its low risk of contamination, cost-effectiveness, possibility of simultaneous assays (multiplex), rapid execution and low detection limit, which can reach 1 copy/mL depending on the pathogen being assessed. Despite this, certain pathogens may present latency phases, characterized by low or no viral replication9, making it impossible to detect them using molecular methods and therefore guaranteeing a reliable negative diagnosis, requiring the association of an immunological diagnostic method, such as the Indirect ELISA and Luminex xMAP, which are capable of detecting specific antibodies produced against the pathogen of interest in both singleplex and multiplex formats respectively.After reviewing the literature, we decided to choose 11 pathogens considered important for xenotransplantation by experts from all over the world. The main aim of this project is to build a multiplex molecular (qPCR) and serological (ELISA and LUMINEX) panel comparable to the tests currently considered the gold standard. Biological samples of blood, serum, semen, swabs and tissues from 100 pigs from commercial farms and slaughterhouses will be evaluated. Once the analytical parameters of the tests have been defined, we will draw up operational protocols for testing animals and surfaces, using which we will begin to populate the two pig facilities that are currently under construction. (AU)