# Mass spectrometry-based discovery and diagnostic validation of T. cruzi antigens in the urine of congenitally infected Chagas Disease patients

## TL;DR

Researchers discovered T. cruzi antigens in the urine of infants with congenital Chagas disease, offering a non-invasive diagnostic method that could improve detection in low-resource areas.

## Contribution

The study introduces a novel urinary diagnostic test using T. cruzi-specific peptides and antibodies, with one biomarker never used in Chagas disease diagnostics before.

## Key findings

- 198 T. cruzi-specific peptides were identified in infant urine samples.
- Antibodies against trans-sialidase and mucin-associated surface proteins achieved 87.5% sensitivity and 94.7% specificity in diagnosing Chagas disease.
- A lateral flow immunoassay was validated for potential use in low-resource settings.

## Abstract

Caused by the parasite Trypanosoma cruzi, Chagas disease affects an estimated 7 million people globally. Diagnosis of Chagas disease in infants is urgently needed, as early detection allows for more effective treatment and reduced mortality. However, current diagnostics are inappropriate for effective detection in infants due to differences in the mechanism of disease in infants and the infant immune system, as well as lack of diagnostic sensitivity and loss to follow up. Studying peripheral biomarkers in urine can leverage physiological concentration in the bladder to increase yield of proteins secreted by pathogen, infected cells, or antigen processed by immune cells residing in different body sites.

We analyzed the urine of a cohort of infants who were congenitally infected with Chagas disease, using a method including affinity enrichment, mass spectrometry, and bioinformatics analysis to characterize the T. cruzi secreted peptidome. We identified 198 peptides specific for T. cruzi and analyzed them in light of their potential for diagnostic utility. Our protocol revealed that peptides of the hyper-mutating mucin-associated surface protein and trans-sialidase protein families could be identified in patient urine and can serve as diagnostic markers of disease. We developed antibodies against conserved regions of each protein and validated that these antibodies could be used to differentiate the urine of Chagas disease patients (N = 16 cases) from healthy controls (N = 19). By utilizing affinity enrichment sample preprocessing and anti-trans-sialidase and anti-MASP antibodies in tandem, we differentiated cases from controls with 87.5% sensitivity and 94.7% specificity.

Our work suggests that it is possible to detect Trypanosoma cruzi infection directly from a noninvasively collected fluid such as urine. A direct test in urine with this success rate would be well suited for rapid diagnosis in low-resource areas. Further studies to validate this approach are warranted.

There is an urgent need for a sensitive, specific, and rapid test for congenital Chagas disease (CD) infections. A urinary test detecting protein fragments deriving from the causative agent Trypanosoma cruzi would be well-suited to congenital CD diagnostics due to the ease of collecting urine from infants and the potential for highly accurate detection, with minimal false positive and false negative results. Here, we present a pipeline that led to the discovery of many T. cruzi-specific peptides in urine samples collected from infants born to infected mothers in rural Bolivia. We designed antibodies against the most promising biomarkers and validated those antibodies in patient samples. Due to our use of innovative affinity concentration techniques, which allow for the concentration of low-abundance biomarkers and the exclusion of potentially interfering host proteins, we were able to achieve greater than 85% sensitivity and specificity using these biomarkers. One of these biomarkers has never been used in CD diagnostic testing. We also include experimental evidence validating the reagents for a lateral flow immunoassay, which requires minimal training and equipment to perform and therefore has transformative potential to increase access to care for individuals in rural and remote communities, where CD is most common.