Circulating Exovesicles in Sera of Chronic Patients as a Method for Determining Active Parasitism in Chagas Disease

Abstract

Chagas disease, once a neglected disease confined primarily to the Americas, is now recognized as a global health issue. This shift is attributed to migratory movements from endemic to nonendemic regions where the epidemiological risk is limited to transmission through transplacental transmission to newborns of infected mothers or through blood and organ donations from infected individuals. One challenge in managing Chagas disease is the low or nullpresence of bloodborne parasite forms in chronic patients, complicating diagnosis. Additionally, persistent antibodies throughout patients' lives hinder treatment efficacy assessment. In this study, we investigated circulating Trypanosoma cruzi immunocomplexes as biomarkers of parasite presence since EVs are secreted by metabolically active protozoan forms. These immunocomplexes contain parasite antigens recognized by antibodies from two immunosera: one against a total extract of trypomastigote forms of the parasite and another against the signal peptide of MASP proteins, specific to T. cruzi and absent in other Trypanosomatids. Additionally, we evaluated two methods for purifying circulating EVs: the "gold standard" ultracentrifugation and a less equipment-intensive method suitable for diagnostic laboratories, allowing assessment of active protozoan parasitism. Our results suggest that the choice of technique to isolate circulating EVs in serum depends on specific objectives and the technological capabilities of the laboratory. Recognition of these antigens in patient sera confirms the usefulness of these EVs as a confirmatory test for active parasite presence. Moreover, T. cruzi EVs have been detected in PCR negative newborn babies up to 9th months after birth, which would indicate the versatility of these methods to detect active parasitemia.