Abstract Immunodiagnostic tests for detecting dengue virus infections encounter challenges related to cross-reactivity with other related flaviviruses. Our research focuses on the development of a synthetic multiepitope antigen tailored for dengue immunodiagnostics. Selected dengue epitopes involved structural linearity and dissimilarity from the proteomes of Zika and Yellow fever viruses which served for computationally modeling the three-dimensional protein structure, resulting in the design of two proteins: rDME-C and rDME-BR. Both proteins consist of seven epitopes, separated by the GPGPG linker, and a carboxy-terminal 6 × -histidine tag. The molecular weights of the final proteins rDME-C and rDME-BR are 16.83 kDa and 16.80 kDa, respectively, both with an isoelectric point of 6.35. The distinguishing factor between the two proteins lies in the origin of their epitope sequences, where rDME-C is based on the reference dengue proteome, while rDME-BR utilizes sequences from prevalent Dengue genotypes in Brazil from 2008 to 2019. PyMol analysis revealed exposure of epitopes in the secondary structure. Successful expression of the antigens was achieved in soluble form and fluorescence experiments indicated a disordered structure. In subsequent testing, rDME-BR and rDME-C antigens were assessed using an indirect Elisa protocol against Dengue infected serum, previously examined with a commercial diagnostic test. Optimal concentrations for antigens were determined at 10 µg/mL for rDME-BR and 30 µg/mL for rDME-C, with serum dilutions ranging from 1:50 to 1:100. Both antigens effectively detected IgM and IgG antibodies in Dengue fever patients, with rDME-BR exhibiting higher sensitivity. Our in-house test showed a sensitivity of 77.3 % and 82.6 % and a specificity of 89.4 % and 71.4 % for rDME-C and rDEM-BR antigens. No cross-reactivity was observed with serum from Zika-infected mice but with COVID-19 serum samples. Our findings underscore the utility of synthetic biology in crafting Dengue-specific multiepitope proteins and hold promise for precise clinical diagnosis and monitoring responses to emerging Dengue vaccines. crossreactivity cross reactivity flaviviruses immunodiagnostics threedimensional three dimensional structure rDMEC rDME C rDMEBR. rDMEBR BR. BR linker carboxyterminal carboxy terminal histidine tag 1683 16 83 16.8 1680 80 respectively 635 35 6.35 proteome 200 2019 testing 1 µgmL µg mL 3 rDMEC, C, 150 50 1:5 1100. 1100 100. 100 1:100 patients inhouse house 773 77 77. 826 82 82. 894 89 4 89. 714 71 71. rDEMBR rDEM Zikainfected COVID19 COVID 19 COVID-1 samples Denguespecific specific vaccines 168 8 16. 63 6.3 20 201 15 5 1: 110 1:10 7 COVID1 COVID- 6. 2 11 1:1

Objective: To assess the reproducibility of Tronzo and AO/ASIF classifications for transtrochanteric fractures, in order to determine the most appropriate classification for clinical application, and to evaluate the influence of the level of experience of the observers in the agreement between evaluations.Methods: We selected 30 radiographic images of transtrochanteric fractures of the femur, which were presented to two groups of observers, one formed by expert physicians and the other by resident physicians.Results: When evaluated together, Tronzo classification obtained a Kappa value of 0.44. The same classification assessed by the expert group obtained a value of 0.46, while the group of residents' value was 0.44. Evaluating the AO/ASIF classification of the complete pool analysis the value found was 0.42. For the same classification, analyzed by the expert group, obtained a value of 0.41, and by the group of residents, the Kappa value achieved was 0.42. However, when analyzed in its simplified form, the AO/ASIF classification obtained Kappa values of 0.70 (pooled analysis), 0.68 (experts) and 0.72 (residents), considered concurrent.Conclusion: The AO/ASIF simplified classification showed substantial reproducibility and is, therefore, recommended as the most suitable for clinical application. The level of experience of the observers did not influence significantly the agreement between evaluations. Level of Evidence III, Diagnostic Study - Investigating a Diagnostic Test.