Identification of Novel Diagnostic Serum Biomarkers for Chagas' Disease in Asymptomatic Subjects by Mass Spectrometric Profiling

Abstract

More than 10 million people are thought to be infected with Trypanosoma cruzi, primarily in the Americas. The clinical manifestations of Chagas' disease (CD) are variable, but most subjects remain asymptomatic for decades. Only 15 to 30% eventually develop terminal complications. All current diagnostic tests have limitations. New approaches are needed for blood bank screening as well as for improved diagnosis and prognosis. Sera from subjects with asymptomatic CD (n ‫؍‬ 131) were compared to those from uninfected controls (n ‫؍‬ 164) and subjects with other parasitic diseases (n ‫؍‬ 140), using protein array mass spectrometry. To identify biomarkers associated with CD, sera were fractionated by anion-exchange chromatography and bound to two commercial ProteinChip array chemistries: WCX2 and IMAC3. Multiple candidate biomarkers were found in CD sera (3 to 75.4 kDa). Algorithms employing 3 to 5 of these biomarkers achieved up to 100% sensitivity and 98% specificity for CD. The biomarkers most useful for diagnosis were identified and validated. These included MIP1 alpha, C3a anaphylatoxin, and unusually truncated forms of fibronectin, apolipoprotein A1 (ApoA1), and C3. An antipeptide antiserum against the 28.9-kDa C terminus of the fibronectin fragment achieved good specificity (90%) for CD in a Western blot format. We identified full-length ApoA1 (28.1 kDa), the major structural and functional protein component of high-density lipoprotein (HDL), as an important negative biomarker for CD, and relatively little full-length ApoA1 was detected in CD sera. This work provides proof of principle that both platform-dependent (i.e., mass spectrometry-based) and platform-independent (i.e., Western blot) tests can be generated using high-throughput mass profiling.

Advances and challenges towards a vaccine against Chagas disease

“…Nonetheless, ongoing efforts for the identification of additional biomarkers are underway, including electrocardiographic, immunologic and biochemical markers, which may greatly improve and facilitate the monitoring of Chagas disease progression and thus vaccine evaluation. [86][87][88][89][90][91][92] As indicated above, a successful vaccine will need to induce a strong cellular immune response, with the activation of CD8 + cytotoxic T cells in order to effectively control T. cruzi parasites. This requirement places some constraints on the formulations and types of vaccines that may be used, since such a response is harder to induce than a humoral response.…”

Discovery and Characterization of Potential Prognostic Biomarkers for Dengue Hemorrhagic Fever

“…Serum samples were fractionated using the ProteinChip Serum Fractionation kit (Bio-Rad Laboratories) by anion-exchange chromatography and pH gradient as previously described by Ndao. 25 Fractions 1, 5, and 6 were collected and applied to cationic (CM10), metal affinity (IMAC), and hydrophobic (H50) ProteinChip arrays (Bio-Rad Laboratories). The fractions were then washed to remove non-specific binding, and the energy absorbing molecule was applied.…”

“…Cluster data were analyzed with BPS (Bio-Rad Laboratories) as previously described by Ndao 2010. 25 The BPS used the classification and regression tree (CART) method, to identify peaks that best discriminated between groups.…”

Chronic Chagas disease: from basics to laboratory medicine

“…All of these could possibly be utilized to develop laboratory medicine tools that can be used for risk assessment and/or the early diagnosis of Chagas heart disease. Modern platforms, such as mass spectrometric profiling of serum, have been used increasingly for the identification of novel serum markers in Chagas patients [112,113] . Finding such markers in asymptomatic patients and the evaluation of their potential for risk assessment would be a future step.…”