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

Abstract

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.

Fig 1. Antigenic recognition of EVs obtained from sera of patients with CCD by concentrators vs differential centrifugation.

Relative absorbance was calculated as the absorbance values at 490 nm, minus the mean absorbance value of the cut-off value, obtained by the same immunoassays and treatment against EV from individuals without CD. A. Developed with anti-MASP SP immunoserum. B. Developed with anti-T. cruzi immunoserum.

Fig 2. Antigenic recognition by anti-T. cruzi and anti-MASP SP immunosera of EVs obtained by ultracentrifugation from sera of Bolivian patients with CD diagnosed in Spain and classified by symtoms.

The absorbance value at 490 nm is represented on the y-axis and samples from different patients are represented on the x-axis. The absorbance results are the net values after subtracting the cut-off value from the total absorbance A. Patients with CD and indetermined symthoms. B. Patients with CD and cardiac disorder, and with cardiac and gastrointestinal symptoms together. C. Patients with gastrointestinal symptoms.

Fig 3. Absorbance obtained by ELISA with the different serum samples.

Comparison between rural and urban samples of patients diagnosed with CD in the Panamanian population. Black triangles for patients living in rural areas and blue circles for urban patients in Panama City. Red asterisks show sera that were selected for proof-of-concept for Fig 4 used in subsequent experiments with antigen recognition in circulating EVs. The horizontal line represents the cut-off value of the negative sera obtained from the mean of the absorbances of these sera plus three times the standard deviation of the means of these negative sera (mean absorbance of negative sera +3 x SD).

Fig 4. Detection by ELISA of EVs with T. cruzi antigens in the serum of patients whose diagnosis by conventional diagnostic systems was inconclusive.

Red: positive control; yellow: negative control; blue: patients with CCD positive by PCR; Green: patients with CD but negative by rapid test; Brown: patients with CD and negative by WB and Wiener ELISA test; Purple: patients with CD negative by Wiener ELISA test; Orange: patients with CD positive by rapid test and Wiener ELISA but negative by WB.

Fig 5. Trypanosoma cruzi antigenic recognition in EVs obtained from sera of mothers with CD and their children by anti-T. cruzi immunoserum.

The absorbance value at 490 nm is represented on the y-axis and samples from 16 different mothers and their children are represented on the x-axis. The absorbance results are the net values of subtracting the absorbance of the cut-off value from the absorbance obtained from the samples.

Fig 6. Boxplots illustrating the relative absorbance values measured at 490 nm for each IgG subclass isolated from immunocomplexes obtained from patients with CD.

A. Mean values of patients originating from Bolivia with different pathologies. Red: cardiac pathology; Green: indeterminate patients; Blue: gastrointestinal pathology; Orange: combined cardiac plus gastrointestinal symptoms. B. Patients with CD originating from Panama. Mean and SD are represented for each isotype. Red: IgG1; Green: IgG2; Blue: IgG3 and Orange; IgG4.