Chagas Disease Diagnosis with Trypanosoma cruzi-Exclusive Epitopes in GFP
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethical Considerations for the Use of Human Serum
2.2. Serological Samples
2.3. Cloning, Expression, and Purification
2.4. Enzyme-Linked Immunosorbent Assays
2.5. Spot Synthesis Analysis
2.6. Statistics
3. Results
3.1. Design and Production of DxCruziV1
3.2. Design and Production of DxCruziV1
3.3. Conditions for Performing In-House ELISAs
3.4. Epitope Contribution to Signal
3.5. Design, Production, and Performance of DxCruziV2
3.6. Analytical Sensitivity of DxCruziV1 and DxCruziV2
4. Discussion
5. Conclusions
6. Patents
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Epitope | Sequence | Insertion Site a | AA Duplication b | Protein Origin | Refs. | |
---|---|---|---|---|---|---|
DxCruzV1 | 1 | KFAELLEQQKNAQFPGK | 20 | - | KMP11 | [34] |
2 | KAAAAPA c | 74 | KL | TcE | [35,36] | |
3 | KAAIAPA d | 124 | DV | TcE | [35,36] | |
4 | GDKPSPFGQAAAADK | 139 | GT | PEP-2; B13; Ag2 | [37,38,39] | |
5 | KQKAAEATK | 161 | LK | CRA | [40,41,42] | |
6 | AEPKPAEPKS | 178 | TAD | TcD-2 | [36,39] | |
7 | AEPKSAEPKP | 199 | GS | TcD-1 | [36,39] | |
8 | GTSEEGSRGGSSMPS | 215 | DGP | TcLo 1.2 | [36] | |
9 | SPFGQAAAGDK | 238 | EL | PEP-2; B13; Ag2 | [37,38,39] | |
10 | KQRAAEATK | C-term | - | CRA | [40,41,42] | |
DxCruziV2 | 1 | KFAELLEQQKNAQFPGK | 1 | - | KMP11 | [34] |
11 | DSSAHSTPSTPA | 74 | KL | SAPA | [43,44] | |
4 | GDKPSPFGQAAAADK | 139 | GT | PEP-2; B13; Ag2 | [37,38,39] | |
12 | FGQAAAGDKPS | 162 | KLK | 3973 (TcCA-2) | [45] | |
6 | AEPKPAEPKS | 178 | TAD | TcD-2 | [36] | |
13 | PPSGTENNKPATG | 199 | GSGTSWKGS | TSSA | [46] | |
8 | GTSEEGSRGGSSMPS | 215 | DGP | TcLo1.2 | [36] | |
9 | SPFGQAAAGDK | 238 | EL | PEP-2; B13; Ag2 | [37,38,39] | |
3 | KAAIAPA | C-term | GTSWKGD | TcE | [35,36] | |
10 | KQRAAEATK | C-term | - | CRA | [40,41,42] |
Parameter | DxCruziV1 | DxCruziV2 | ||
---|---|---|---|---|
O.D. 405 | >0.2635 | >0.3170 | >0.2600 | >0.3065 |
Sensitivity (95% CI a) | 100.0% (93.98 to 100.0%) | 100.0% (93.98 to 100.0%) | 96.67% (88.64 to 99.41%) | 96.67% (88.64 to 99.41%) |
Specificity (95%CI) | 98.68% (93.98 to 100.0%) | 100.0% (95.19 to 100.0%) | 98.68% (92.92 to 99.93%) | 100.0% (95.19 to 100.0%) |
Kappa Index | 0.97 | 1.00 | 0.94 | 0.96 |
Likelihood Ratio | 76.0 | - | 73.47 | - |
False Positive | 2.7% | 0.0% | 1.3% | 1.3% |
False Negative | 0.0% | 0.0% | 3.3% | 5.0% |
PPV b | 96.8 | 100.0 | 98.3 | 98.3 |
NPV c | 100.0 | 100.0 | 97.4 | 96.1 |
Concordance | 98.5% | 100.0% | 97.8% | 97.0% |
Non-Concordance | 1.5% | 0.0 | 2.2% | 3.0% |
Area Under Curve | 1 | 0.998 | ||
Std. Error | 0 | 0.001661 | ||
95% CI | 1.00 to 1.00 | 0.995 to 1.00 | ||
p value | <0.0001 | <0.0001 |
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Durans, A.d.M.; Napoleão-Pêgo, P.; Reis, F.C.G.; Dias, E.R.; Machado, L.E.S.F.; Lechuga, G.C.; Junqueira, A.C.V.; De-Simone, S.G.; Provance, D.W., Jr. Chagas Disease Diagnosis with Trypanosoma cruzi-Exclusive Epitopes in GFP. Vaccines 2024, 12, 1029. https://doi.org/10.3390/vaccines12091029
Durans AdM, Napoleão-Pêgo P, Reis FCG, Dias ER, Machado LESF, Lechuga GC, Junqueira ACV, De-Simone SG, Provance DW Jr. Chagas Disease Diagnosis with Trypanosoma cruzi-Exclusive Epitopes in GFP. Vaccines. 2024; 12(9):1029. https://doi.org/10.3390/vaccines12091029
Chicago/Turabian StyleDurans, Andressa da M., Paloma Napoleão-Pêgo, Flavia C. G. Reis, Evandro R. Dias, Luciana E. S. F. Machado, Guilherme C. Lechuga, Angela C. V. Junqueira, Salvatore G. De-Simone, and David W. Provance, Jr. 2024. "Chagas Disease Diagnosis with Trypanosoma cruzi-Exclusive Epitopes in GFP" Vaccines 12, no. 9: 1029. https://doi.org/10.3390/vaccines12091029