Development of a New Lateral Flow Assay Based on IBMP-8.1 and IBMP-8.4 Chimeric Antigens to Diagnose Chagas Disease

Abstract

Despite several available methodologies for Chagas disease (CD) serological screening, the main limitation of chronic CD diagnosis is the lack of effective tools for large-scale screening and point-of-care diagnosis to be used in different CD epidemiological scenarios. Taking into account that developing such a diagnostic tool will significantly improve the ability to identify CD carriers, we aimed at performing a proof-of-concept study (phase I study) to assess the use of these proteins in a point-of-care platform using serum samples from different geographical settings of Brazil and distinct clinical presentations. The diagnostic accuracy study was conducted on a panel of two WHO International Standards (IS) and 14 sera from T. cruzi-positive and 16 from T. cruzi-negative individuals. The results obtained with the test strips were converted to digital images, allowing quantitative comparison expressed as a relative band intensity ratio (RBI). The diagnostic potential and performance were also determined. Regardless of the geographical origin or clinical presentation, all sera with T. cruzi antibodies returned positive both for IBMP-8.1 and IBMP-8.4 chimeric antigens. The area under the ROC curve (AUC) values was 100% for both antigens, demonstrating an outstanding overall diagnostic accuracy (100%). Based on the data, we believe that the lateral flow assays based on these antigens are promising methodologies for screening CD.

Figure 1

Lateral flow assay (LFA) device. (a) Schematic illustration of the composition of the LFA strip for the detection of IgG anti-Trypanosoma cruzi in a clinical sample employing the IBMP-8.4 and IBMP-8.1 chimeric antigens; and photographic image of an LFA opened device after loading T. cruzi specific IgG antibodies. (b) Schemes of the results expected for valid and invalid results yielded by LFA.

Figure 2

Flowchart illustrating the study design in conformity with the Standards for Reporting of Diagnostic Accuracy Studies (STARD) guidelines. Public domain digital map was freely obtained from the Brazilian Institute of Geography and Statistics (IBGE) cartographic database in shapefile format (.shp), which was subsequently reformatted and analyzed using QGIS version 3.10 (Geographic Information System, Open Source Geospatial Foundation Project. http://qgis.osgeo.org).

Figure 3

Photographic images of the lateral flow assays (LFA) strips after loading T. cruzi-positive (upper panel) or negative (lower panel) samples. BA (Bahia); CA (cardiac form); CD (cardio-digestive form); GO (Goiás); IN (indeterminate form); MG (Minas Gerais); MX (Mexico); N (T. cruzi-negative result); P1 (T. cruzi-positive result characterized by 41.0 ≥ RBI ≤ 60.0); P2 (T. cruzi-positive result characterized by 60.0 > RBI ≤ 80.0); P3 (T. cruzi-positive result characterized by 80.0 > RBI ≤ 100); PE (Pernambuco); SP (São Paulo); RBI (relative band intensity).

Figure 4

Assay performance parameters were obtained with serum from noninfected and T. cruzi-infected individuals. The cut-off value is represented by the dotted lines. Horizontal lines and numbers for each group of results represent the geometric means (±95% CI). Acc (accuracy); AUC (area under curve); ROC curve (receiver operating characteristic curve); Sen (sensitivity); Spe (specificity).