Circulating extracellular vesicles in sera of chronic patients as a method for determining active parasitism in Chagas disease.

BACKGROUND: Chagas disease, once restricted mainly to the Americas, Chagas disease has become a global health problem due to migration from endemic to non-endemic areas. In non-endemic regions, transmission is limited to vertical transmission from infected mothers to newborns or through blood and organ donations. A major challenge in the management of the disease lies in the diagnosis of chronic cases, as blood-borne parasites are often absent and antibodies persist for life, complicating the evaluation of treatment. METHODOLOGY AND MAIN FINDINGS: This study investigates whether detection of circulating extracellular vesicles (EVs) or their immunocomplexes with host IgGs in the serum of chronic patients with Chagas disease could serve as diagnostic tools and biomarkers of the active presence of the parasite. This method may prove valuable in cases where parasitaemia and other diagnostic tests are inconclusive, especially for assessing treatment efficacy and confirming mother-to-child transmission. Together with exovesicle purification by ultracentrifugation, which is the 'gold standard', an affordable and simplified method for the isolation of EVs or immunocomplexes was tested for use in less well-equipped diagnostic laboratories. EV detection was performed by enzyme-linked immunosorbent assay (ELISA) targeting Trypanosoma cruzi antigens. Positive results were demonstrated in Bolivian patients in Spain, covering asymptomatic and symptomatic cases (cardiac, gastrointestinal or both). The study also examined infected mothers and their newborns. These findings were further confirmed in Panamanian patients with inconclusive diagnostic results. Moreover, host IgG isotypes that formed immunocomplexes with parasite exovsicles were identified, with IgG2 and IgG4 being predominant. CONCLUSIONS: Our results confirm the usefulness of circulating EVs and their immunocomplexes as markers of metabolically active T. cruzi in chronic infections without detectable parasitaemia, as well as their efficacy in confirming vertical transmission and in cases of inconclusive diagnostic tests.