Validation of Apolipoprotein A-1 and Fibronectin Fragments as Markers of Parasitological Cure for Congenital Chagas Disease in Children Treated With Benznidazole

Abstract

Background: No reliable tests or validated biomarkers exist to ensure parasitological cure following treatment of Chagas disease (CD) patients chronically infected with Trypanosoma cruzi. As seroreversion, the only marker of cure, happens more quickly in children, we investigated the correlation between previously identified biomarkers and seroreversion in children.

Methods: Thirty CD children (age 1 month to 10 years) diagnosed as T. cruzi positive (time point S0) were treated with benznidazole (BZ) 5-8 mg/kg/d for 60 days. At least 2 serological tests were used to evaluate treatment efficacy from the end of treatment (S1) until seroreversion (S2). Thirty children (age 1 month to 10 years) and 15 adults were used as healthy controls (HCs). Immunoblot and a proteomic-based assay were used to validate previously identified fragments of apolipoprotein A-1 (ApoA1) and fibronectin (FBN) as CD biomarkers.

Results: Correlation between seroreversion and absence of ApoA1 and FBN fragments by immunoblot was observed in 30/30 (100%) and 29/30 (96.6%) CD children, respectively. ApoA1 and FBN fragments were absent at the end of BZ treatment in 20/30 (66.6%) and 16/30 (53.3%) children, respectively. Absence of fragments in serum profiles was confirmed by mass spectrometry. Using intact protein analysis, a 28 109-Da protein identified as full-length ApoA1 by tandem mass spectrometry was detected in HC serum samples.

Conclusions: These data confirm that ApoA1 and FBN fragments can discriminate between healthy and T. cruzi-infected samples. Correlation with seroreversion was shown for the first time; results suggest predictive capacity potentially superior to serology, making them potentially useful as surrogate biomarkers.

Keywords: Chagas disease; apolipoprotein A-1; biomarkers; children; fibronectin; test of cure.

Figure 1.

Immunoblot analysis of ApoA1 (24.7) and FBN (28.9) fragments in CD and HC. Each antiserum detects expected bands in a CD child but not in child or adult HCs. The specificity of the rabbit anti-ApoA1 (24.7) serum (A) and rabbit anti-FBN (28) (B) for the corresponding fragments is demonstrated by their inability to detect native full-length proteins, ApoA1 (~28.1 KDa) and FBN (~220 KDa), while reacting with 6xHis tag recombinants of the 24.7-KDa ApoA1 and 28.9-KDa FBN (migration at ~35 KDa) fragments, respectively. Abbreviations: ApoA1, apolipoprotein A-1; CD, Chagas disease; HC, healthy control; FBN, fibronectin; FL, full-length; R, recombinants.

Figure 2.

Immunoblot analysis of apolipoprotein A-1 (24.7) and fibronectin (28.9) fragments in serum samples from Chagas disease (CD) children at different time points. Serum from CD children age >1 year and younger were collected at diagnosis (S0), end of treatment (S1), and at seronegative conversion (S2). Immunoblot analysis was performed on 1 uL of serum using polyclonal antibodies against neo-peptides PALEDL and PFTDV from APOA1 24.7-KDa and FBN 28.9-KDa fragments, respectively.

Figure 3.

A, Volcano plot for all proteins identified in serum sasmples of Chagas disease (CD) and healthy control (HC) children. Mass of 28 109 Da, eluted at 38.5 minutes of the liquid chromatography gradient, (red point) is significatively upregulated in HC compared with CD. B, Tandem mass spectrometry spectra of 28 109 Da mass precursor peak and Biotools results that confirm the apolipoprotein A-1 identity.

Figure 4.

Representative mass spectrometry spectra of biomarkers 24.7 (A) and 28.9 (B) for samples from Chagas children patients treated with benznidazole. Proteins of 24 756 and 28 786 Da are present at diagnosis (S0), the intensity of 24 756 Da is reduced after treatment (S1), and both biomarkers are no longer detected at seroreversion (S2). A peak of 28 078 Da corresponding to apolipoprotein A-1 (~28.1 KDa) is detected at seroreversion.

Figure 4.

Representative mass spectrometry spectra of biomarkers 24.7 (A) and 28.9 (B) for samples from Chagas children patients treated with benznidazole. Proteins of 24 756 and 28 786 Da are present at diagnosis (S0), the intensity of 24 756 Da is reduced after treatment (S1), and both biomarkers are no longer detected at seroreversion (S2). A peak of 28 078 Da corresponding to apolipoprotein A-1 (~28.1 KDa) is detected at seroreversion.

Figure 5.

Mass spectrometry spectra of biomarkers 24.7 (A) and 28.9 (B) for children’s samples that show negative immunoblot results at the end of benznidazole (BZ) treatment (S1). Proteins of 24 758 and 28 803 Da are present at diagnosis (S0) but are undetected at the end of BZ treatment (S1) and seroconversion (S2); instead, proteins corresponding to full-length apolipoprotein A-1 (~28.1 KDa) are detected at S1 and S2.