Discovery and validation of de novo generated endogenous biomarkers for the diagnosis of Chagas disease

Abstract

Chagas disease (CD) remains a debilitating neglected tropical disease and a major public health concern in endemic and non-endemic areas, affecting more than 7 million people worldwide. CD is caused by Trypanosoma cruzi, a protozoan parasite mostly transmitted by triatomine insect vectors in endemic regions of Latin America. CD progresses in three clinical phases: acute (ACD), indeterminate/latent, and chronic (CCD) phases. Fast and accurate diagnosis of CD is paramount for timely treatment, because the two principal treatment options, benznidazole (BZ) and nifurtimox (Nfx), often have unsatisfactory/undeterminate cure rates in chronically infected patients. Clinical presentation of Chagas disease is unspecific, necessitating the need for parasitological, serological and molecular diagnostic methods. Parasitological methods include direct microscopic detection of circulating parasites, or indirectly by haemocultures or xenodiagnoses. These approaches offer direct evidence of parasites in patients but are limited by low parasitaemia in the chronic phase, operator dependence and laboriousness. Molecular tools including qPCR and LAMP offer high specificity for the diagnosis of CD, but are often expensive, unamendable in low-resource settings, low sensitivity in chronic phase disease, and require trained clinical personnel. Immunological tests, that detect anti-T. cruzi IgG antibodies in circulation, include enzyme-linked immunosorbent assay (ELISA), indirect haemagglutination assay (IHA), indirect immunofluorescence assay (IIF), trypomastigote excreted secreted antigens (TESA) blot, chemiluminescent magnetic immunoassays (CMIA) and Rapid diagnostic tests (RDTs). These tests are mostly applied in chronic phase disease, and often have high sensitivities, but are limited by being labor intensive, cross-reactivity with other parasite antigens, and operator-dependent interpretation of results. The bottlenecks faced in the diagnosis of Chagas disease, and the need for early and accurate detection of T. cruzi infection to facilitate timely treatment when cure rates are favorable during the acute phase (ACD), necessitate the urgent discovery and validation of novel, specific, sensitive and robust diagnostic biomarkers. To address these limitations, the proposed study will employ mass spectrometry-based approaches to discover novel biomarkers in patient biofluids from T. cruzi-infected patients and healthy controls in endemic regions from the state of Pará, Brazil. To meet this aim, the proposed study will be formulated into three main areas of research: 1) Analysis of acute CD patients and healthy controls using MALDI-TOF MS and computational tools developed by our group to identify CD-specific protein profiles in biofluids. To identify the specific biomarkers, LC-MS/MS analysis will be performed; 2) Determination of differential protease activities in serum from ACD-infected and non-infected patients using mass spectrometry-based approaches. The biomolecules detected in this objective will be protein fragments generated by the action of circulating proteases released by the parasites or endogenously present in the host; 3) applying the novel computational platform developed by our group, termed PhyloQuant, to differentially abundant proteins detected in the sera of ACD patient and healthy control. The optimization and application of this tool for ACD biomarker discovery will be an important tool for prioritizing biomarkers in other diseases. The abundance of protein and protein fragments identified in this study will be validated using orthogonal techniques such as western blotting or ELISA.The expected results from the proposed study will be the identification and validation of Chagas disease phase-specific protein profiles, and the identification of specific de novo generated endogenous biomarkers for the diagnosis of Chagas disease. (AU)