RESUMO
We developed a protein to rapidly and accurately diagnose Chagas disease, a life-threatening illness identified by the WHO as a critical worldwide public health risk. Limitations in present day serological tests are complicating the current health situation and contributing to most infected persons being unaware of their condition and therefore untreated. To improve diagnostic testing, we developed an immunological mimic of the etiological agent, Trypanosoma cruzi, by combining ten pathogen-specific epitopes within the beta-barrel protein structure of Thermal Green Protein. The resulting multi-epitope protein, DxCruziV3, displayed high specificity and sensitivity as the antibody capture reagent in an ELISA platform with an analytical sensitivity that exceeds WHO recommendations. Within an immunochromatographic platform, DxCruziV3 showed excellent performance for the point of application diagnosis in a region endemic for multiple diseases, the municipality of Barcelos in the state of Amazonas, Brazil. In total, 167 individuals were rapidly tested using whole blood from a finger stick. As recommended by the Brazilian Ministry of Health, venous blood samples were laboratory tested by conventional assays for comparison. Test results suggest utilizing DxCruziV3 in different assay platforms can confidently diagnose chronic infections by T. cruzi. Rapid and more accurate results will benefit everyone but will have the most noticeable impact in resource-limited rural areas where the disease is endemic.
Assuntos
Doença de Chagas , Ensaio de Imunoadsorção Enzimática , Epitopos , Testes Sorológicos , Trypanosoma cruzi , Doença de Chagas/diagnóstico , Doença de Chagas/sangue , Doença de Chagas/imunologia , Humanos , Ensaio de Imunoadsorção Enzimática/métodos , Trypanosoma cruzi/imunologia , Testes Sorológicos/métodos , Epitopos/imunologia , Doença Crônica , Masculino , Sensibilidade e Especificidade , Feminino , Adulto , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Pessoa de Meia-Idade , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/sangue , Brasil/epidemiologiaRESUMO
Serological tests are critical tools in the fight against infectious disease. They detect antibodies produced during an adaptive immune response against a pathogen with an immunological reagent, whose antibody binding characteristics define the specificity and sensitivity of the assay. While pathogen proteins have conveniently served as reagents, their performance is limited by the natural grouping of specific and non-specific antibody binding sites, epitopes. An attractive solution is to build synthetic proteins that only contains pathogen-specific epitopes, which could theoretically reach 100% specificity. However, the genesis of de novo proteins remains a challenge. To address the uncertainty of producing a synthetic protein, we have repurposed the beta barrel of fluorescent proteins into a receptacle that can receive several epitope sequences without compromising its ability to be expressed. Here, two versions of a multiepitope protein were built using the receptacle that differ by their grouping of epitopes specific to the parasite Trypanosoma cruzi, the causative agent for Chagas disease. An evaluation of their performance as the capture reagent in ELISAs showed near-complete agreement with recommended diagnostic protocols. The results suggest that a single assay could be developed for the diagnosis of Chagas disease and that this approach could be applied to other diseases.