Ethics statement

For the study of Bolivian patients residing in Spain, we have the permissions of the following Ethics Committees: Ethics Committees of HUyP-La Fe with the numbers HUyP-La Fe, Valencia, Spain (2016/0866), and of the University of Granada, Human University of the Granada Ethical Committee, Spain, with number n°: 672/CEIH/2018.

All participating patients received an ‘informed consent’ document to be signed, which is included in the supplementary material. The document includes a section for the “clinical study in children”, where the notification to the Public Prosecutor’s Office is included, as required by Spanish legislation for the participation of minors, and which must be signed by the parents or legal guardians of the minors.

For the study of the patients from Panama, permission was obtained from the Ethics Committee of the University of Panama, Faculty of Medicine with number 2015-310V1 and from the Human Ethics Committee of the University of Granada with number 672/CEIH/2018. All Panamanian patient participants received an informed consent document which they were required to sign in accordance with Panamanian regulations.

Study populations

In this study, two populations of patients with CCD from Latin America were examined.

The first population comprised 92 patients of Bolivian origin who currently reside in Spain. These individuals live in the Spanish city of Valencia, where they were diagnosed and underwent medical follow-up at the Hospital Universitario y Politécnico La Fe (HUyP-La Fe) Valencia, Spain. The Bolivian samples included 92 patients who underwent screening tests, applying three serological assays: i) LiaisonXL murex (Diasorin), ii) rapid test (SD Bioline Chagas Ab Rapid, Abbott 49FK10), and iii) IFA kit (Trinity Biotech). All these tests, as mentioned earlier, are routinely applied for the diagnosis of individuals suspected of being affected by CCD at the HUyP-La Fe. Of the 92 Bolivian patients, 63 were adults with CCD, comprising 49 (77.8%) females and 14 (22.2%) males. Additionally, there were 16 newborns from mothers with CD and 13 CD-negative individuals, selected as controls.

The 63 patients with positive immunological tests or PCR (Lozano et al 2023) were summoned to the Infectious Diseases Unit, Internal Medicine Department, for control and monitoring of CD. A physical examination, an electrocardiogram, a chest x-ray, an echocardiogram, a barium enema and esophagogram were performed and it was registered whether they presented any cardiac or digestive symptoms at the hospital electronic medical chart. Patients with possible cardiac involvement also underwent cardiac magnetic resonance imaging and 24-hour Holter monitoring. Of these of the adult patients, 24 had indeterminate symptoms, including 16 pregnant women. There were 20 patients with cardiac involvement, 14 with gastrointestinal pathology, and 5 with both cardiac and gastrointestinal symptoms. In the case of the 16 newborns, samples were analyzed by PCR at birth, repeated at one month and at nine months of age.

The second group, and as a proof of concept of the results obtained with patients diagnosed in Spain, consisted of 106 individualized sera of CCD patients, coming from Panama a country considered endemic for CD since 1930. The samples had been used in a previous work by our research group [50] and in which the DTU of the parasites that produced the infection had been typed. Sampling was conducted in both rural areas, where patients have continuous contact with vector insects, and urban areas within Panama City. Informed consent procedures, surveys, and blood sample collection were carried out for all patients who voluntarily chose to participate in the study. Of the studied Panamanian population, a total of 106 potential CD patients were analyzed, with 78 out of the 106 (73.58%) residing in the rural community of Chararé, located in the mountainous region of the Las Margaritas, Chepo district, Panama province (coordinates 9.243640, -79.059162). Screening was conducted using three serological tests: i) rapid test (SD Bioline Chagas Ab Rapid, Abbott 49FK10); ii) WB using the methodology previously described by Saldaña et al. (1995) [51]; iii) ELISA Chagatest (Wiener lab 1293257) [50]. Only individuals who tested positive in two out of these three tests were considered positive. Out of the initial 106 individuals, 53 were identified as positive (33 from rural areas and 20 from urban areas) and were used to validate the presented diagnostic methods. A selection of 25 sera randomly selected, from the individuals previously diagnosed as positive in Panama [50] by screening with at least two of the serological tests described above and clinically evaluated in the hospitals of Chepo and Santo Tomas, after an electrocardiographic examination and thoracic radiography and clinically classified as Chagas positive. In all cases, negative parasitization by Trypanosoma rangeli and Leishmania ssp. was evaluated, which in the case of Panamanian leishmaniasis is of cutaneous pathology. In order to calculate the cut-off value of the absorbances obtained from the circulating Evs in the sera of the patients. Fifty sera were used as shown in S3 Table, from individuals negative by immunological and PCR tests for CD, from the same geographical region. Of these sera, five showed syphilis pathology and two showed leprosy pathology. None of these cases showed infection by Leishmania ssp. or T. rangeli.

For the validation of immunocomplexes detection after dissociation of the immunoglobulins present in the sera of CCD patients, a total of 117 sera from both patient populations were used. This included 92 sera from the population diagnosed at HUyP-La Fe, and a selection of 25 sera from the 53 individuals previously diagnosed as positive in Panama through screening with the three serological tests described above. This selection included two sera negative for ELISA Chagatest, two negative for WB, two negative for the rapid test, one negative for both ELISA Chagatest and WB, 16 positives for all three screening tests, plus a negative reference serum and an existing positive reference serum in our laboratory’s serum bank.

Immunological tests used for Chagas disease patient diagnosis

As previously mentioned, the immunodiagnostic tests employed for diagnosing patient populations varied based on their availability at the hospitals where the patients were recruited and analyzed.

For patients of Bolivian origin diagnosed at HUyP-La Fe, analysis was conducted using the LiaisonXL murex kit (Diasorin). This kit employs a chemiluminescence immunoassay with recombinant antigenic proteins (multi-antigen). The methodology followed was in accordance with the manufacturer’s recommendations. As a second diagnostic test, an immunofluorescence assay (IFA) kit (Trinity Biotech) was used, following the manufacturer’s guidelines.

For the diagnosis and confirmation of the Panamanian patients, the initial test used was the rapid test (SD Bioline Chagas Ab Rapid, Abbott 49FK10). To conduct the assay, 100 μL of serum were deposited into the sample well along with 50 μL of assay buffer. Test interpretation was performed visually after a 15-minute incubation period at room temperature.

Another diagnostic test used for CD diagnosis in this population was a commercial ELISA test for antibodies against T. cruzi, specifically the ELISA Chagatest (Wiener lab 1293257). The assay was carried out and validated following the manufacturer’s instructions; accordingly, serum samples were diluted to a concentration of 1:20.

All serum samples from Central American patients underwent an antigen recognition test for the parasite by patient immunoglobulins using WB, following the methodology described by Saldaña et al. (1995) [51] and subsequently published by Ledezma et al. (2020) [50]. For diagnostic use, the transferred strips were incubated with patient serum (1:100) for 2 hours. Subsequent treatment after the washes was done with peroxidase-conjugated rabbit secondary antibodies (Dako, anti-Human IgA, IgG, IgM, Kappa, Lambda/HRP, ref: P0212) diluted 1:700 in PBS, for 1 hour, as previously described [33].

Isolation of immunocomplexes by ultracentrifugation “Gold Standard” and dissociation of purified immunocomplexes (Ig-EVs)

The purification of circulating immunocomplexes (Ig-EVs) in the serum of patients was carried out following methods previously described by Díaz-Lozano et al. (2017) [29], and Lozano et al. (2023) [52] through a mixed procedure of filtration through 0.45 μm filters followed by differential ultracentrifugation at 110.000 xg for 2h in in microcentrifuge tubes (Hitachi No 1508) at 110,000 × g for 2 hours at 4 °C in a CP100NX centrifuge (Hitachi Koki, Tokyo, Japan) with a fixed-angle rotor P70A.

After this centrifugation stage, the pellets containing the immunocomplexes were washed three times by ultracentrifugation in sterile filtered PBS and evaluated through nanoparticle tracking analysis (NTA) and transmission electron microscopy, as described in Lozano et al. (2023) [52] and Cornet-Gomez et al 2023 [53].

In order to separate the EVs present in the sera from the immunoglobulins forming the immunocomplexes, the pellet containing the immunocomplexes was resuspended in 90 μl of PBS containing a cocktail of protease inhibitors without EDTA (Roche, ref: 11836170001). Subsequently, to the suspension containing the immunocomplexes, 650 μl of 0.1 M glycine-HCl at pH 4 were added and incubated for 15 minutes at room temperature. This suspension in glycine-HCl pH 4 buffer was ultracentrifuged again at 100,000 x g for 1 hour to separate circulating EVs in the pellet and the immunoglobulin solution that forms the immunocomplexes in the supernatant. The supernatant was aliquoted and pH-neutralized with Tris-HCl Buffer, pH 10, Antigen Retriever (Sigma T6455) containing 0.1% Glycerol, frozen, and kept at -20 °C until use for IgGs purification by affinity chromatography using Protein G and subsequent determination of the isotype, as described later. The pellet obtained from ultracentrifugation was resuspended in PBS, centrifuged again at 100,000 x g, the supernatant removed, and resuspended in 80 μl of 0.1 M bicarbonate buffer (pH 9.6) containing protease inhibitors without EDTA (Roche, ref: 11836170001).

Isolation and concentration of EVs through filtration with centrifugal concentrators

As an alternative method, for the concentration of EVs and immunocomplexes present in serum in order to avoid using the ultracentrigugation techniques described above, which may be unaffordable for hospital and health center laboratories, the method using protein concentrators previously described by Orrego et al. (2021) [54] and Ramírez et al. (2018) [55] was followed, with some modifications.

Briefly, patient serum (1 ml) was diluted with 5 ml of ultrafiltered PBS. Subsequently, centrifugation at 1,500 x g for 10 minutes was conducted, and the resulting supernatant was filtered through a pore size of 0.45 μm. Following this initial filtration and centrifugation, the filtered supernatant underwent a further centrifugation step at 3,500 × g for 20 minutes. The final supernatant, diluted with PBS was applied to Vivaspin protein concentrator (Sartorius Lab Instrument, Goettingen, Germany) with a separation cut-off size of 100K (100,000 MWCO), which were centrifuged at 6,000 × g for 1 hour at 4 °C. The retained volume in each concentrator was collected, aliquoted, and stored at -80 °C until use.

Determination of the size of EVs and purified immunocomplexes

The hydrodynamic size distribution of the purified immunocomplexes obtained by either method described above was measured by NTA (Nanoparticle Tracking Analysis), using an instrument equipped with a sample chamber, a 405-nm laser, and a high-sensitivity complementary metal-oxide-semiconductor (CMOS) camera. The samples were diluted in 0.22 μm filtered PBS up to 1 ml and then loaded into the chamber. Three 60 s videos, in Brownian mode, were recorded and analyzed for each sample with NTA 2.3 image-analysis software (NanoSight Ltd., Amesbury, UK). The mean size distribution was calculated as a mean of three independent size distributions. This methodology follows the procedures previously described by Retana-Moreira et al. (2021) [48] and by Lozano et al. (2023) [52] and by Cornet-Gomez et al. (2023) [53].

Use of animals for the Production of antisera and authorization by the animal welfare and ethics committee

The use of animals for obtaining antisera was carried out in accordance with the guidelines set forth in the Spanish Government Regulation (Royal Decree RD1201/05) and the European Union Directive (European Directive 2010/63/EU). It was approved by the Ethics Committee of the University of Granada and by the Regional Government authorities of Andalusia (Junta de Andalucía) with the number ES1802100000038 in 2017.

Preparation of polyclonal antibodies against T. cruzi

Three four-week-old male Wistar rats were intraperitoneally immunized with 20 μg of a total extract from T. cruzi Pan4 trypomastigotes per dose, combined with Freund’s adjuvant, to produce polyclonal anti-T. cruzi antibodies. The parasite extract was derived from 10⁹ trypomastigotes obtained from cell cultures, which were previously washed and concentrated by centrifugation, following the procedure described by Cornet et al. (2023) [53].

Antibody titers in serum samples were determined on a weekly basis after the first two immunizations using an indirect ELISA. At the end of the immunization period (8 weeks), the animals were euthanized in an isoflurane atmosphere. Whole blood samples were obtained by cardiac puncture.

To design the synthetic peptide corresponding to the consensus sequence of the signal peptide (SP) of MASP proteins, the methodology described by Díaz-Lozano et al. (2017) [29] was followed. A consensus sequence (MAMMMTGRVLLVCALCVLWSVAADG) (S3 Fig) was used, which was synthesized by LifeTein (USA, LLC) with four branches joined by lysine residues.

The production of polyclonal antibodies against the synthetic peptide corresponding to the signal peptide sequence of MASP proteins was carried out in three four-week-old male Wistar rats with 100 μg of the MASP SP peptide per dose, respectively. Before the first immunization step, a blood extraction was performed in all cases to obtain preimmune control serum. Antibody titers of anti-MASP SP and anti-T. cruzi extract sera were verified by an indirect ELISA in multiple well microtiter plates (Nunc, Thermo Fisher) coated with 5 μg of the antigen/well in 0.1 M bicarbonate coating buffer (pH 9.6). Sera with titers greater than 1:6,400 were selected and stored at -80 °C, diluted 1:1 with glycerol (Molecular Biology grade, Sigma) until use, and were referred to as anti-MASP SP antisera or anti-T. cruzi total antisera.

Electrophoretic confirmation of Trypanosoma cruzi antigens present in circulating EVs in the plasma of patients

In order to recognize the T. cruzi antigens present in the EVs isolated from the plasma of patients affected by Chagas disease, the western blot methodology described in Retana Moreira et al [26] was followed, for which the purified EV proteins were precipitated in acetone at -20 °C overnight. They were centrifuged at 13,000 x g for 10 min at 4 °C and washed twice with cold acetone. After the acetone residue was removed, the precipitated proteins were quantified using the Micro BCA Protein Assay Kit (Thermo Scientific, ref: 23235). 30 μg of these proteins were loaded into the wells of 12% SDS-PAGE gels after electrophoresis and subsequent transfer to PVDF membranes (BioRad, Hercules, CA, USA). The membranes were immersed in blocking buffer (PBST plus 4% skimmed milk). Incubated at 4°C for 12h with a 1:1000 dilution of polyclonal anti-T. cruzi antibody, the membranes were washed and incubated with a secondary antibody anti rat (1:10000) peroxidase-conjugated goat antibody (Sigma-Aldrich, ref: A9037) for 1h at room temperature. Detected bands were visualized using Clarity ECL Western Substrate (BioRad, Hercules, CA, USA) on a ChemiDoc Imaging system (BioRad, Hercules, CA, USA).

Antigenic recognition by ELISA by anti-MASP SP or anti-T. cruzi immunosera from EVs isolated from patient sera

For ELISA assays, Nunc 96 multi-well plates (Thermo Fisher Scientific) with a volume of 100 μl per well were coated with a concentration of 5 μg/μl of proteins from a lysate of EVs, in RIPA buffer, isolated from each serum sample in 100mM carbonate/bicarbonate buffer (pH 9.6). The protein concentration was determined using the Micro BCA Protein Assay Kit (Thermo Scientific, ref: 23235), following the instructions. Plates were incubated under shaking for 8 hours at 4 °C. After adsorption, the plates were twice washed with PBST to remove unbound antigens.

Subsequently, 250 μl of a freshly prepared blocking solution were added and incubated at 4 °C under shaking overnight. The plates were washed again with PBST. Then, 100 μl of rat T. cruzi primary serum (1:2000) or anti-MASP SP (sera with titters higher than 1:6400) diluted 1:500 in PBS were added to each well, and the plates were incubated for 2 hours under shaking at room temperature. After antibody interaction, the plates were washed at least three times in PBST, and 100 μl of peroxidase conjugated polyclonal goat anti-rat IgG (Sigma-Aldrich, ref: A9037) at a dilution of 1:1000 in PBS were added, followed by incubation at room temperature under shaking for 1 hour.

Following incubation with the secondary antibodies, the plates were washed four times, and O-phenyl-diaminobenzidine plus 30% H₂O₂ (1 μl/ml) (Sigma-Aldrich) was added to 0.05 M phosphate-citrate buffer, pH 5.0, as a peroxidase substrate. The plates were further incubated for 15 minutes at 27 °C. The reaction was halted with a solution of 0.1 M 2 N H₂SO₄, and absorbance was measured at 492 nm using an ELISA Multiskan Spectrum reader (Thermo Fisher Scientific).

To determine the cut-off value of the EVs, these were individually purified from the plasma of Chagas disease-negative 13 control sera of the same origin and geographic region that were challenged by ELISA with anti-MASP SP and anti-T. cruzi immunosera, as described in the previous paragraph. And where, three negative sera from Spain, two positive for Leishmania infantun and one positive for leprosy, diseases in which cross-reactions with CD have been described, were included.

The cut-off value was calculated as the average OD at 492 nm plus three times the standard deviation value of the samples (mean of the OD + 3 x SD).

Isotyping of immunoglobulin G (IgG) forming immunocomplexes with EVs isolated from CD-Positive plasma

IgGs were separated from the immunocomplexes by treatment with 0.1 M glycine-HCl at pH 4 and subsequent ultracentrifugation as described above. The IgGs were purified by chromatography using Protein G HP SpinTrap/Ab Spin Trap columns (GE Healthcare Life Sciences, 28-9031-34), following the manufacturer’s instructions. Briefly, after removing the storage buffer from the columns, the column was equilibrated with 600 μL of binding buffer composed of 20 mM sodium phosphate (Sigma-Aldrich, 255793) in PBS. Washed twice with 600 μL of binding buffer to remove those sample components not bound to protein G from the column, the columns were eluted by centrifugation for 2 min at 100xg in 40 0ul of elution buffer composed of (0.1 M Glycine-HCl, pH 3.0). The columns were eluted in 2 ml vials containing 15 μL of basic pH buffer (Tris-HCl buffer, pH 10, Antigen Retriever). Elution as in the previous steps was performed by centrifugation at 100 × g for 2 minutes, repeated twice, resulting in a final eluate volume of 800 μL for each of the immunocomplex samples from each of the sera.

Once the purified IgGs were obtained, they were quantified using the Micro BCA Protein Assay Kit (Thermo Scientific, ref: 23235).

Isotyping of different IgG isotypes, as well as immunoglobulin subclasses, was performed via ELISA following the previously described methodology. Primary antibodies included IgG immunoglobulins (1:1,000) from rat (anti-human IgG2a ThermoFisher), mouse (anti-human IgG1 and anti-human IgG2 of Sigma, anti-human IgG2b of Biomedicals, and anti-human IgG3 and anti-human IgG4-HRP of Abcam) were incubated for 1 hour at room temperature under shaking. As a secondary antibody labeled with HRP peroxidase (except for IgG4, which is already labeled with HRP), a 1:1,000 dilution of anti-rat (Sigma-Aldrich, ref: A9037) or anti-mouse (Dako, ref: P0447) was added and incubated for 1 hour at room temperature with gentle stirring.

Washes were performed as described above with PBST, and 100 μl of peroxidase as substrate and incubated for 20 min at room temperature under shaking in the dark. Finally, 50 μl of 3M HCl stop solution in distilled water were added, and the reading was carried out at 492 nm in a MultiskanSpectrum spectrophotometer (Thermo Fisher Scientific).

Statistical analysis

The Shapiro test was used for testing normality of the distribution of the data. Normal distributed data are expressed as mean (± standard deviation) and were compared with the ANOVA and Tukey test. Nonparametric data are expressed as median (interquartile range) and were compared with the Mann-Whitney U test and the Kruskal-Wallis test. Nonparametric related data was studied with the Wilcoxon signed-rank test and the Bonferroni-corrected significance level method was applied.

Statistical analyses were performed with Rstudio and a p < 0.05 was considered significant.

Sample selection. CCD patients of Bolivian origin living in Spain

Of the patients of Bolivian origin diagnosed at HUyP-La Fe, all were positive in the three immunological techniques mentioned (LiaisonXL murex (Diasorin), rapid test (SD Bioline Chagas Ab Rapid, Abbott 49FK10), IFA kit (Trinity Biotech); hence they were used in the subsequent studies (S1 Table). Of these, 63 were randomly selected from various situations and pathologies, including indeterminate symptoms, cardiac pathology, gastrointestinal disturbances, and both cardiac and gastrointestinal disturbances. Additionally, 16 pregnant women with CD and their newborns were included (summary in S2 Table).

Ultracentrifugation vs Filtration for the detection of T. cruzi Antigens in EVs from Sera of CCD patients

The protein concentrator method used was designed, as already indicated, to facilitate the purification and concentration of EVs from serum or plasma in order to facilitate instrumentally and technologically the collection of EVs from serum. We compared the use of protein concentrators to purify EVs from plasma with the gold standard method for the purification of EVs (ultracentrifugation).

The results obtained from the characterization of the NTA purified EVs with both methods are shown in S1A and S1B Fig and in Table 1.

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Table 1. Results of the determination of the size and amount of EV protein obtained by the two purification procedures.

(*) (p-value <0.00001).

https://doi.org/10.1371/journal.pntd.0012356.t001

With the method in which protein concentrators were used, as shown in the Table 1, larger peaks were obtained, possibly aggregates of the EVs with each other (S1A and S1B Fig). The total protein concentration was statistically higher when the ultracentrifugation method was used for purification (27.9 ± 10.8 μg/μl) compared to the alternative filtration method (9.5 ± 8 μg/μl) (p-value <0.00001) (S2 Fig).

In order to test the antigenic recognition in EVs purified by protein concentrators and the gold standard of EV purification by the two immunisera (anti MAM Sp and anti Tc (total)), 24 individual serum samples were randomly selected from a total of 63 clinically characterized positive samples for CD from patients residing in Spain.

Among these 24 samples individually selected samples, there were 7 pregnant women. Of the 24 samples, (S1 Table), 14 patients had indeterminate symptoms, while 5 had gastrointestinal symptoms and 5 had cardiac pathology. Each sample was treated by both ultracentrifugation-ultrafiltration and concentrator purification to separate circulating vesicles, as described in Materials and Methods.

Purification of EVs by ultracentrifugation and detection with anti-MASP SP detected those 21 patients had relative absorbance values above the cut-off threshold, while for the remaining three, the absorbance was close to 0. This same pattern was replicated when the EVs were purified by the filtration method (Fig 1A).

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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.

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Employing the anti-T. cruzi for EV detection, 23 patients showed absorbance values above the cut-off threshold when EVs were purified through both ultracentrifugation and filtration (Fig 1B).

For the same EV isolation method, no significant differences were found between the two immunosera used. The Wilcoxon rank sum test yielded W = 208.5 and a p-value >0.05 for the ultracentrifugation method, and W = 374 with a p-value >0.05 for the filtration method.

For both markers, absorbance values were significantly higher when applying the ultracentrifugation technique to purify EVs from serum samples of CCD patients compared to purification via the filtration technique. Specifically, anti-T. cruzi (W = 94, p-value <0.05) and anti-MASP SP (W = 99, p-value <0.05) showed ELISA elevated absorbance levels. These findings were derived using the Wilcoxon rank sum test with continuity correction, as the variables did not adhere to a normal distribution. This suggests a consistent trend favoring the ultracentrifugation method over filtration in both markers (Fig 1A and 1B).

Determination of antigenic recognition in EVs from Sera of CCD patients resident in Spain by Immunosera, anti-T. cruzi and anti-MASP SP

The patients were categorized into groups based on their symptoms: indeterminate symptoms (24 total patients), cardiac pathology (20 total patients), gastrointestinal symptoms (14 total patients), and those presenting with both cardiac and gastrointestinal pathologies (5 total patients).

Of the 24 samples from indeterminate patients, EVs reacted in 23 (95.8%) samples against anti-T. cruzi. The absorbance levels were under the cut-off value in a 42-year-old man (number 7), who had a previous positive PCR in 2010 and was treated, reactivity of EVs obtained to both immunosera gave absorbance values below the cut-off value (Fig 2A and Table 2).

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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.

https://doi.org/10.1371/journal.pntd.0012356.g002

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Table 2. A summary of the results obtained with anti-T. cruzi and anti-MASP SP in the different clinical situations.

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When anti-MASP SP immunoserum was used against EVs, 20 of the 24 indeterminate patients samples (83%) had absorbances higher than the cut-off value. The absorbance levels were lower than the cut-off value in four patients, number 7 and 13 had positive PCRs and was treated, number 17 and 53 had a negative PCR and number 53 was a pregnant woman (Fig 2A and Table 2).

Of the 39 symptomatic CD patients, 35 (89.7%) samples were positive with anti-T. cruzi, and 32 (82,1%) samples with anti-MASP SP (Table 2).

Two cardiac patients (patients numbers 16 and 18), 1 gastrointestinal (patient number 45) and 1 cardiac and gastrointestinal patient (patient number 25) presented lower absorbance levels with anti-T. cruzi than the cut-off values (Fig 2B and 2C).

Two cardiac patients (patients number 16 and 18), 3 gastrointestinal patients (patients number 43, 45 and 46) and 2 cardiac and gastrointestinal patients (patients number 25 and 27) had lower absorbance levels with anti-MASP SP than the cut-off values (Fig 2B and 2C).

The results of antigen recognition in all patient groups, (cardiac, gastrointestinal and cardiac plus gastrointestinal pathologies) shows that the detection of EVs was more effective with anti-T. cruzi immunoserum than with anti-MASP SP (Mann-Whitney U 993.5, p < 0.00001) for all the patients with different disorders.

When we evaluated the antigenic recognition obtained in EVs using anti-T. cruzi, significant differences were observed in the absorbances obtained depending on the pathology shown by the patients (Kruskal-Wallis test, chi-square = 14.536, df = 3, p-value = 0.00226). The group of patients with indeterminate symptoms showed significantly higher absorbance values compared to the absorbance obtained with circulating EVs from the cardiac and gastrointestinal patient groups (Bonferroni-corrected significance level method, p-value <0.05). However, this trend was not statistically significant when the anti-MASP SP immunoserum was used (Kruskal-Wallis test, chi-square = 6.1322, df = 3, p-value = 0.1054).

Sample selection. Patients in Endemic areas, Panama

The rapid tests applied allowed us to obtain quick and reliable results, capable of having a first sweep of the population under study, mainly in the rural area where health resources are scarce, since their results are qualitative or semiquantitative and the samples do not require any type of equipment, testing system or specialized refrigeration. In the rural patients of the Chararé community, 87.8% (29/33) were positive, while 75% (15/20) of the urban patients analyzed presented positive results in the rapid test (S1 Table).

A second commercial ELISA test (Wiener lab) was conducted in 53 chronic Chagas patients. Among them, 54% (29/53) tested positive, while 6% (3/53) were classified as clearly inconclusive due to their absorbance values falling within the "grey" zone or at the cut-off value (cut-off = 0.3). Additionally, 40% (21/53) tested negative. Serum samples from a rural population displayed a 42% (14/33) positivity rate for the aforementioned ELISA test method, while sera from urban patients selected from the hospital showed a 75% (15/20) positivity rate. All serum samples were evaluated in triplicate, and the results were recorded (Fig 3 and Table 3).

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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).

https://doi.org/10.1371/journal.pntd.0012356.g003

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Table 3. A summary of the results obtained with commercial tests and our proof of concept test.

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The WB analysis reveals distinct antigenic bands in positive sera (25, 30, 45, 52, 70 kDa), while the remaining bands are regarded as nonspecific for CD diagnosis. S4A Fig illustrates results from patients in urban areas, where 90% (18/20) tested positive, and 10% (2/20) yielded inconclusive results. In contrast, patients from the rural community of Chararé (S4B Fig), exhibited an 84% (28/33) positivity rate, with 15% (5/33) showing indeterminate results (Table 3).

Detection of circulating parasite EVs from the sera of CCD patients from an endemic country (Panama) by anti-T. cruzi immunoserum. proof of concept

As proof of concept to evaluate the utility of using circulating EVs as an indicator of the active presence of the parasite in sera where immunological diagnostic systems had been inconclusive, we decided to purify EVs from a total of 25 (16 positive for all techniques; 2 negative for the rapid test; 2 negative for the Wiener ELISA test; 2 negative for WB and one negative for WB and ELISA) as indicated in the text and caption of Fig 4 including a positive control and a negative control. This includes all diagnostic situations of these patient sera of Central American CCD patients from Panama that were in such a situation despite testing PCR positive. Once the circulating immunocomplexes from the serum were purified and any accompanying IgGs that could form immunocomplexes were eliminated, these EVs were confronted with anti-T. cruzi immunoserum. Negative controls consisted of EVs extracted from panel of 50 serum samples from immunologically negative individuals who lacked symptomatology after medical evaluation of these individual sera from the same geographical region as described in S3 Table and above, but lacking the disease, and treated with a similar procedure as the sera under study.

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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.

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Fig 4 shows that anti-T. cruzi antisera recognize antigens in EVs isolated from the sera of 23 patients from Panama who presented inconclusive or negative in commercial diagnostic tests for CD, including WB results. In all cases, the absorbance obtained was higher than the cut-off value obtained from the pool of sera from non-infected individuals.

Detection of circulating parasite EVs in sera of pregnant Bolivian Women and their infants

The study on the presence of EVs in pregnant women who tested positive for CD and sequentially tested for reactivity of EVs in their babies at 1 month and subsequently at 9 months after birth revealed that the mean absorbance value at 490 nm for anti-T. cruzi in the babies at the first month was 2.08 (95% CI: 1.77–2.27), whereas at 9 months, the obtained global result was 1.24 (95% CI, 0.74–1.61). Furthermore, a significant correlation was found with the Spearman test between absorbance values at 1 month and 9 months (Rho 0.72; p-value = 0.009) (Fig 5).

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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.

https://doi.org/10.1371/journal.pntd.0012356.g005

In samples corresponding to children number 5 and 13, the absorbance values were equal to or below the cut-off value using the immunoserum against total T. cruzi antigen at nine months after birth (Fig 5).

Samples from patients number 7, 9, 11 and 12 at 9 months of age could not be collected as they did not return to the hospital for follow-up of the mother or child.

Sample number 16 corresponds to a child who received treatment two months after birth due to the positive PCR of the mother during pregnancy and subsequently of the child one month after birth. The treatment consisted of Benznidazole, administered orally in three doses for 60 days, 10 mg/kg per day for the child. The mother received the same drug administered orally in two doses for 60 days at 5–7.5 mg/kg per day. At the beginning of the treatment, lactation was interrupted. The absorbance after recognition of the EVs by the anti-T. cruzi immunosorbent serum at 9 months after birth was very low in this child, treated and with interrupted breastfeeding.

Children in samples 5 and 13 showed significantly lower absorbance levels (similar or lower than the cut-off value at 9 months) between samples taken at 1 and 9 months of age in contrast to samples from children who still showed elevated absorbance at 9 months, (1, 2, 3, 4, 6, 8) although they experienced lower absorbance values compared to those obtained from mothers or at 1 month after birth, except for the child in sample 8, where the absorbance at 1 month was higher than that of the mother. The HSD Tukey test revealed a p-value <0.001 (Fig 5).

In sample 10, the values of circulating antigens in the parasite EVs, were maintained from the first month to the ninth month (HSD Tukey test revealed a p-value >0.05), while in the rest of the samples the absorbance values decreased with respect to those obtained at one month after birth.

The samples from mothers 1, 2, 14, and 16 tested positives in the PCR. While mothers 2, 7, 9, 11, 14, 15 and 16 were treated, the children of these mothers tested negative in the PCR at 2 months after birth. Except for number 16, who tested positive in the PCR and received treatment as indicated above (S2 Table). S1 Table shows the peculiarities of each of the cases in detail.

Study of IgG subclasses in the immunocomplexes from Chronic CD patients (Bolivia and Panama) by ELISA

After purifying circulating immunocomplexes (IgGs-EVs) from the serum of CD patients using the ultracentrifugation method described above, we isolated the IgG antibodies that are part of the immunocomplexes to characterize the subclasses of IgGs forming them.

The results of the Bolivian patients in Spain are represented in Fig 6A. In this analysis, significant differences were observed in the absorbance values between the different IgG subclasses when compared with the different pathologies (ANOVA, Isotypes: p-value <0.0001; Pathology: p-value <0. 0001). Specifically, IgG2 and IgG4 isotypes exhibited statistically higher levels (Tukey HSD test with 95% CI: diff = 12.11, p-value = 0 and diff = 9.36, p-value = 0, respectively), while IgG3 did not show significant differences compared to the other subclasses (Tukey HSD test, p-value >0.05).

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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.

https://doi.org/10.1371/journal.pntd.0012356.g006

When comparing Bolivian patient groups categorized as indeterminate and symptomatic (including cardiac, gastrointestinal, and combined cardiac plus gastrointestinal pathologies) via ANOVA (p-value <0.05), significant differences were found for each isotype. In all cases, the indeterminate patient group exhibited significantly higher levels compared to the diagnosed and pathologically affected patient group, as indicated by the following statistics IgG1 (Tukey test with 95% CI: diff = -1.946763, p-value >0.0001); IgG2 (Tukey test with 95% CI: diff = -6.996506, p-value >0.0001); IgG3 (Tukey test with 95% CI: diff = -1.387083, p-value >0.01) and IgG4 (Tukey test with 95% CI: diff = -4.088365, p-value >0.001).

All the patients from Panama graphed in Fig 6B were diagnosed with cardiac conditions. Interestingly, significant differences were found between all variables (IgG1, IgG2, IgG3, and IgG4) when compared to each other, following adjustment for multiple comparisons using the ANOVA method (p-value <2e-16 ***). The mean values of the IgG2 and IgG4 isotypes were significantly higher than those of IgG1 and IgG3 (Tukey HSD test, p-value >0.05). However, the absorbance values of IgG2 and IgG4 were found to be similar for these patients (Tukey HSD test, p-value >0.05).