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Background  The complexity of the eukaryotic parasite  Trypanosoma (T . ) cruzi  manifests in its highly dynamic genome, multi-host life cycle, progressive morphologies and immune-evasion mechanisms. Accurate determination of infection or Chagas’ disease activity and prognosis continues to challenge researchers. We hypothesized that a diagnostic platform with higher ligand complexity than previously employed may hold value.    Methodology  We applied the ImmunoSignature Technology (IST) for the detection of  T .  cruzi -specific antibodies among healthy blood donors. IST is based on capturing the information in an individual’s antibody repertoire by exposing their peripheral blood to a library of >100,000 position-addressable, chemically-diverse peptides.    Principal findings  Initially, samples from two Chagas cohorts declared positive or negative by bank testing were studied. With the first cohort, library-peptides displaying differential binding signals between  T .  cruzi  sero-states were used to train an algorithm. A classifier was fixed and tested against the training-independent second cohort to determine assay performance. Next, samples from a mixed cohort of donors declared positive for Chagas, hepatitis B, hepatitis C or West Nile virus were assayed on the same library. Signals were used to train a single algorithm that distinguished all four disease states. As a binary test, the accuracy of predicting  T .  cruzi  seropositivity by IST was similar, perhaps modestly reduced, relative to conventional ELISAs. However, the results indicate that information beyond determination of seropositivity may have been captured. These include the identification of cohort subclasses, the simultaneous detection and discerning of other diseases, and the discovery of putative new antigens.    Conclusions & significance  The central outcome of this study established IST as a reliable approach for specific determination of  T .  cruzi  seropositivity versus disease-free individuals or those with other diseases. Its potential contribution for monitoring and controlling Chagas lies in IST’s delivery of higher resolution immune-state readouts than obtained with currently-used technologies. Despite the complexity of the ligand presentation and large quantitative readouts, performing an IST test is simple, scalable and reproducible.

An ImmunoSignature test distinguishes Trypanosoma cruzi, hepatitis B, hepatitis C and West Nile virus seropositivity among asymptomatic blood donors