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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.

Introdução/Objetivo: Em 2003, os primeiros casos de transmissão do vírus monkeypox (MPXV) fora do continente Africano foram confirmados em um surto nos EUA. Desde então, casos esporádicos em viajantes retornados da África foram descritos. No ano de 2022, a mpox se disseminou rapidamente por diferentes países e foi declarada emergência de saúde pública de importância internacional pela OMS. O vírus monkeypox (MPXV) apresenta manifestações clínicas similares à varíola humana, porém de menor intensidade, usualmente lesões cutâneas, de evolução sincrônica, associadas ou não a sintomas sistêmicos. Neste estudo buscamos caracterizar o perfil clínico-epidemiológico de casos de mpox no surto atual e investigar a ocorrência da doença em grupos populacionais com menor visibilidade inicial. Metodologia: Estudo de corte transversal realizado pelo Núcleo de Enfrentamento e Estudos de Doenças Infecciosas Emergentes e Reemergentes (NEEDIER - UFRJ), onde foram incluídos pacientes com quadro suspeito de mpox do estado Rio de janeiro investigados na UFRJ de 01 de junho a 31 de dezembro de 2022. O diagnóstico foi realizado por PCR, os dados clínico-epidemiológicos foram obtidos na plataforma (REDCap) e analisados por meio do software R(versão-4.2.2). O estudo foi aprovado pelo CEP-HUCFF (CAAE: 62281722.5.0000.5257). Resultados: Foram incluídos 2919 pacientes que possuíam informações na plataforma REDCap, 787 (27%) dos quais foram diagnosticados com mpox. Dentre os positivos, a mediana da idade foi de 33 anos, 725 (92%) pertenciam ao sexo masculino, 430 (55%) se identificavam como homens que fazem sexo com homens e 284 (36%) viviam com HIV. As manifestações clínicas mais frequentes nos casos positivos foram: lesões cutâneas 676 (86%), febre 464 (59%), linfadenopatia 365 (46%), e cefaleia 340 (43%). Houve forte associação de sintomas, como lesões anogenitais (OR = 3,8, p valor < 0,001), proctite (OR= 5,96, p valor < 0,001), edema peniano (OR = 3,68, p valor < 0,001) e linfadenopatias (OR= 4,38, p valor = 0,001), com a infecção por MPXV na coorte total. Foram detectados 62 (8%) mulheres e 34 (4%) menores de 18 anos entre os casos positivos. Conclusão: Foram observadas mudanças em relação a apresentação clássica da doença, padrão de transmissão e acometimento de mulheres e crianças. Baixo limiar de suspeição clínica e testagem precoce favorecem o diagnóstico rápido, permitindo a intervenção efetiva na cadeia de transmissão e o manejo adequado da doença.

ABSTRACT The COVID-19 pandemic has led to the commercialization of many antigen-based rapid diagnostic tests (Ag-RDTs), requiring independent evaluations. This report describes the clinical evaluation of the Novel Coronavirus 2019-nCoV Antigen Test (Colloidal Gold) (Beijing Hotgen Biotech Co., Ltd.), at two sites within Brazil and one in the United Kingdom. The collected samples (446 nasal swabs from Brazil and 246 nasopharyngeal samples from the UK) were analyzed by the Ag-RDT and compared to reverse transcription-quantitative PCR (RT-qPCR). Analytical evaluation of the Ag-RDT was performed using direct culture supernatants of SARS-CoV-2 strains from the wild-type (B.1), Alpha (B.1.1.7), Delta (B.1.617.2), Gamma (P.1), and Omicron (B.1.1.529) lineages. An overall sensitivity and specificity of 88.2% (95% confidence interval [CI], 81.3 to 93.3) and 100.0% (95% CI, 99.1 to 100.0), respectively, were obtained for the Brazilian and UK cohorts. The analytical limit of detection was determined as 1.0 × 103 PFU/mL (Alpha), 2.5 × 102 PFU/mL (Delta), 2.5 × 103 PFU/mL (Gamma), and 1.0 × 103 PFU/mL (Omicron), giving a viral copy equivalent of approximately 2.1 × 104 copies/mL, 9.0 × 105 copies/mL, 1.7 × 106 copies/mL, and 1.8 × 105 copies/mL for the Ag-RDT, respectively. Overall, while a higher sensitivity was claimed by the manufacturers than that found in this study, this evaluation finds that the Ag-RDT meets the WHO minimum performance requirements for sensitivity and specificity of COVID-19 Ag-RDTs. This study illustrates the comparative performance of the Hotgen Ag-RDT across two global settings and considers the different approaches in evaluation methods. IMPORTANCE Since the beginning of the SARS-CoV-2 pandemic, we have witnessed growing numbers of antigen rapid diagnostic tests (Ag-RDTs) being brought to market. In the United Kingdom, this was somewhat controlled indirectly as the government offered free tests from a small number of companies. However, as this has now ceased, individuals are responsible for their own acquisition of test kits. Similarly in Brazil, as of January 2022, pharmacies and other health care retailers are permitted to sell Ag-RDTs directly to the community. Many of these Ag-RDTs have not been externally evaluated, and results are not readily available to the public. Thus, there is now a need for a transparent evaluation of Ag-RDTs with both analytical and clinical evaluation. We present an independent review of the Novel Coronavirus 2019-nCoV Antigen Test (Colloidal Gold) (Beijing Hotgen Biotech Co., Ltd.), at two sites within Brazil and one in the United Kingdom.

ABSTRACT Community testing is a crucial tool for the early identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission control. The emergence of the highly mutated Omicron variant (B.1.1.529) raised concerns about its primary site of replication, impacting sample collection and its detectability by rapid antigen tests. We tested the performance of the Panbio antigen rapid diagnostic test (Ag-RDT) using nasal and oral specimens for COVID-19 diagnosis in 192 symptomatic individuals, with quantitative reverse transcription-PCR (RT-qPCR) of nasopharyngeal samples as a control. Variant of concern (VOC) investigation was performed with the 4Plex SARS-CoV-2 screening kit. The SARS-CoV-2 positivity rate was 66.2%, with 99% of the positive samples showing an amplification profile consistent with that of the Omicron variant. Nasal Ag-RDT showed higher sensitivity (89%) than oral (12.6%) Ag-RDT. Our data showed good performance of the Ag-RDT in a pandemic scenario dominated by the Omicron VOC. Furthermore, our data also demonstrated that the Panbio COVID-19 antigen rapid diagnostic test does not provide good sensitivity with oral swabs for Omicron Ag-RDT detection. IMPORTANCE This study showed that the antigen rapid test for COVID19 worked fine using nasal swabs when it was utilized in patients infected with the Omicron variant, showing a concordance with PCR in 93% of patients tested. The nasal swab yielded more reliable results than the oral swab when an antigen rapid diagnosis test (the Panbio COVID-19 antigen rapid diagnostic test) was used in patients infected with the Omicron variant.

BACKGROUND The high demand for adequate material for the gold standard reverse transcription real-time polymerase chain reaction (RT-qPCR)-based diagnosis imposed by the Coronavirus disease 2019 (COVID-19) pandemic, combined with the inherent contamination risks for healthcare workers during nasopharyngeal swab (NP) sample collection and the discomfort it causes patients, brought the need to identify alternative specimens suitable for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). OBJECTIVES The aim of this work was to compare saliva and gingival fluid swabs to NP swabs as specimens for RT-qPCR-based SARS-CoV-2 diagnosis. METHODS We compared gingival fluid swabs (n = 158) and saliva (n = 207) to the rayon-tipped NP swabs obtained from mild-symptomatic and asymptomatic subjects as specimens for RT-qPCR for SARS-CoV-2 detection. FINDINGS When compared to NP swabs, gingival fluid swabs had a concordance rate of 15.4% among positive samples, zero among inconclusive, and 100% among negative ones. For saliva samples, the concordance rate was 67.6% among positive samples, 42.9% among inconclusive, and 96.8% among negative ones. However, the concordance rate between saliva and NP swabs was higher (96.9%) within samples with lower cycle threshold (Ct) values (Ct > 10 ≤ 25). MAIN CONCLUSIONS Our data suggests that whereas gingival fluid swabs are not substitutes for NP swabs, saliva might be considered whenever NP swabs are not available or recommended.

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.