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BackgroundThe coronavirus disease (COVID-19) pandemic has led to an unprecedented public health crisis. Insufficient testing continues to limit the effectiveness of the global response to the COVID-19 pandemic. Molecular testing methods such as reverse transcriptase polymerase chain reaction (RT-PCR) continue to be highly centralized and are a sub-optimal option for population surveillance. Rapid antigen tests (Ag-RDTs) offer multiple benefits including low costs, high flexibility to conduct tests in a wide variety of settings, and faster return of results. Recently, self-test Ag-RDTs (STs) have gained approval in several markets and offer the possibility to expand testing, reaching at-risk populations. While STs have the potential to assist the COVID-19 response, test result integrity, reporting, and appropriate linkage to care continue to hinder the widespread implementation of self-testing programs.MethodsThis protocol presents a mixed-methods pragmatic trial (ISRCTN91602092) to better understand the feasibility of self-testing as part of a contact tracing strategy within the Brazilian public health system. Approximately 604 close contacts of 150 index cases testing positive for COVID-19 will be enrolled. Close contacts will be randomized to either serial (daily) self-testing over a 10-day follow-up period or a more traditional approach to contact tracing with a professional Ag-RDT at one time point post-exposure. Usability workshops and focus group discussions will also be conducted.DiscussionThis study protocol presents a comprehensive plan to assess the effectiveness, operational feasibility, and stakeholder preferences of a serial self-testing strategy for contact tracing within the Brazilian public health system. Our results will contribute to better understanding of the feasibility of a self-testing strategy within the public sector. Potential risks and limitations are discussed. Our findings will have important implications as governments continue working to mitigate the impact of COVID-19, particularly in the context of where to direct limited resources for testing and healthcare infrastructure.

The relationship between N-antigen concentration and viral load within a specimen and across different specimens is essential for interpretation of rapid diagnostic tests (RDT) clinical performance in different use cases. A prospective study was conducted in Porto Velho, Brazil, to investigate RDT performance in different specimen types as a function of the correlation between antigen concentration and viral load. The study included 214 close contacts with recent exposures to confirmed cases, aged 12 years and older and with various levels of vaccination. Antigen concentration was measured in nasopharyngeal swab (NPS), anterior nares swab (ANS), and saliva specimens. Reverse transcriptase (RT)–PCR was conducted on the NPS and saliva specimens, and two RDTs were conducted on ANS and one on saliva. Antigen concentration correlated with viral load when measured in the same specimen type but not across specimen types. Antigen levels were higher in symptomatic cases compared to asymptomatic/oligosymptomatic cases and lower in saliva compared to NPS and ANS samples. Discordant results between the RDTs conducted on ANS and the RT-PCR on NPS were resolved by antigen concentration values. The analytical limit-of-detection of RDTs can be used to predict the performance of the tests in populations for which the antigen concentration is known. The antigen dynamics across different sample types observed in SARS-CoV-2 disease progression support use of RDTs in nasal samples. Given lower antigen concentrations in saliva, tests using saliva is expected to require improved analytical sensitivity to achieve clinical sensitivity similar to testing of nasal samples.

Real-time PCR (qPCR) is a remarkably sensitive and precise technique that allows for amplifying minute amounts of nucleic acid targets from a multitude of samples. It has been extensively used in many research areas and achieved industrial application in fields such as human diagnostics and trait selection in crops of genetically modified organisms (GMO) crops. However, qPCR is not an error-proof technique. Mixing all reagents into a single master mix subsequently distributed onto 96 wells of a regular qPCR plate might lead to operator mistakes such as incorrect mixing of reagents or inaccurate dispensing into the wells. Here, a technique called gelification is presented, whereby most of the water present in the master mix is substituted by reagents that form a sol-gel mixture when submitted to a vacuum. As a result, qPCR reagents are effectively preserved for a few weeks at room temperature or a few months at 2-8

Background Point-of-care and decentralized testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to inform public health responses. Performance evaluations in priority use cases such as contact tracing can highlight trade-offs in test selection and testing strategies. Methods A prospective diagnostic accuracy study was conducted among close contacts of coronavirus disease 2019 (COVID-19) cases in Brazil. Two anterior nares swabs (ANS), a nasopharyngeal swab (NPS), and saliva were collected at all visits. Vaccination history and symptoms were assessed. Household contacts were followed longitudinally. Three rapid antigen tests and 1 molecular method were evaluated for usability and performance against reference reverse-transcription polymerase chain reaction (RT-PCR) on nasopharyngeal swab specimens. Results Fifty index cases and 214 contacts (64 household) were enrolled. Sixty-five contacts were RT-PCR positive during ≥1 visit. Vaccination did not influence viral load. Gamma variants were most prevalent; Delta variants emerged increasingly during implementation. The overall sensitivity of evaluated tests ranged from 33% to 76%. Performance was higher among symptomatic cases and those with cycle threshold (Ct) values 70% and almost 90% after 4 days. Conclusions The near-immediate time to results for antigen tests significantly offsets lower analytical sensitivity in settings where RT-PCR results are delayed or unavailable.

BackgroundPoint-of-care and decentralized testing for SARS-CoV-2 is critical to inform public health responses. Performance evaluations in priority use cases such as contact tracing can highlight trade-offs in test selection and testing strategies.MethodsA prospective diagnostic accuracy study was conducted among close contacts of COVID-19 cases in Brazil. Two anterior nares swabs (ANS), a nasopharyngeal swab (NPS), and saliva were collected at all visits. Vaccination history and symptoms were assessed. Household contacts were followed longitudinally. Three rapid antigen tests and one molecular method were evaluated for usability and performance against reference RT-PCR on NPS.ResultsFifty index cases and 214 contacts (64 household) were enrolled. Sixty-five contacts were RT-PCR positive during at least one visit. Vaccination did not influence viral load. Gamma variants were most prevalent; Delta emerged increasingly during implementation. Overall sensitivity of evaluated tests ranged from 33%–76%. Performance was higher among symptomatic cases and cases with Ct70% and almost 90% after four days.ConclusionsThe near immediate time-to-result for antigen tests significantly offsets lower analytical sensitivity in settings where RT-PCR results are delayed or unavailable.

Rapid and specific diagnosis of malaria remains one of the main strategies to fight the disease. The diagnosis is made primarily by the simple and low-cost thick drop technique, considered the gold standard test. However, the requirement for good quality microscopes and well-trained personnel often lead to inaccurate diagnosis, especially in cases of mixed infections or low parasitemia. Although PCR-based tests can help in these situations, this technique requires large and sensitive equipments, being unsuitable for point of care (POC) settings. A myriad of POC diagnostic tests have being developed in the last years, relying on molecular methods but also on novel strategies. New platforms, miniaturization techniques, and multiplexing possibilities promise great potential to improve disease diagnostics through fast and accurate detection of cases, even at remote places. Here, we will address the main POC strategies developed for the diagnosis of malaria, highlighting their strengths and weakness as POC applications.

Trachoma, caused by the bacteria Chlamydia trachomatis, is the world leading infectious cause of blindness. The disease is associated with poor living conditions, especially in developing countries. In these countries, diagnosis is usually based on clinical evaluation, although laboratory confirmation is necessary. Serological-based tests for trachoma laboratory confirmation are cheaper than molecular-based tests, but the later are more sensitive and specific. Among the available molecular tests, qPCR has the best cost-benefit. With this in mind, the present study developed a new duplex qPCR reaction, which concomitantly detects C. trachomatis cryptic plasmid and the human 18S rRNA gene, as an internal reaction control. The new qPCR was validated using 50 previously qPCR-characterized samples for trachoma infection, and showed 95% specificity and 100% sensitivity, with an estimated LOD95 of 600 ag/µL. Next, 50 samples from an endemic area (Marajó, Pará) and 12 from a non-endemic area (Curitiba, Paraná) were investigated using direct immunofluorescence assay (DFA) or the new duplex qPCR. Among the 50 endemic samples, three were positive by clinical evaluation (6%), 18 by DFA (36%) and 48 by qPCR (96%). All samples positive by the clinic evaluation were also positive by qPCR. From the 18 DFA-positives, qPCR identified 16 as positives as well. On the other hand, 32 samples that were DFA-negative due to the low number of elementary bodies (

Orientador: Anibal E. Vercesi Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas Resumo: O presente trabalho está dividido em duas partes: a reconstituição funcional de uma proteína desacopladora de Arabidopsis thaZiana e a medida da expressão e da atividade de uma proteína desacopladora em resposta a diversos estresses abióticos. A expressão do gene AtPUMPl em Saccharomyces cerevisae e Escherichia coZi permitiu a confirmação de que este gene realmente codifica uma proteína desacoplado~ capaz de dissipar energia do gradiente eletroquímico de Ir. Num ambiente termoneutro (28°C), células expressando a AtPUMPl cresceram mais lentamente que células controle, enquanto sob baixa temperatura (14 °C) a taxa de crescimento de ambas as células foi semelhante. Quando expressa em um sistema procariótico e reconstituída em vesículas lipídicas, a AtPUMPl mostrou características cinéticas semelhantes às descritas para as outras proteínas desacopladoras. O transporte de Ir foi induzido por diferentes ácidos graxos, de uma maneira dependente do tamanho da cadeia de carbonos e do número de insaturações na mesma. Este transporte de Ir foi inibido por nucleotídeos de purina, sendo que mudanças no pH do meio externo alteraram a afinidade da AtPUMPl por nucleotídeos. Através dos experimentos de reconstituição, verificamos que as constantes de afinidade e velocidade máxima de transporte (Km e V máx) são similares às obtidas para outras proteínas desacopladoras vegetais e também para as UCP2 e 3. A expressão e a atividade da proteína desacopladora também foi estudada em situações de estresse abiótico, como excesso de sal, temperatura baixa e atmosfera hiperoxigenada, situações que sabidamente geram estresse oxidativo. Em coleóptiles de milho, a expressão do mRNA para a proteína desacopladora aumenta durante o estresse salino e permanece inalterada durante o estresse por mo. Entretanto, a expressão da proteína e a disponibilidade de substratos aumentaram significativamente nos dois casos, sugerindo uma regulação pós-transcripcional para este gene. Observamos ainda que mitocôndrias isoladas de coleóptiles de milho submetidos a estresse salino e por frio possuem uma razão ADP/O diminuída, que retoma para níveis controle na presença de BSA ou GTP. Em situações de aumento intracelular de Ca2+, observamos também que ativadores da proteína desacopladora inibem a geração mitocondrial de EAOs e que seus inibidores aumentam esta geração. Considerando os resuhadqs aqui apresentados, podemos, portanto, concluir que a AtPUMPl é uma verdadeira proteína desacopladora, capaz de afetar o crescimento celular, induzindo um desacoplamento mediado por ácidos graxos e inibido por nucleotídeos de purina. É possível sugerir ainda que a proteína desacopladora desempenhe um papel importante em situações de estresse abiótico, diminuindo a geração mitocondrial de EAOs de fonna a proteger o organismo do estresse oxidativo resultante Abstract: The present work is divided in two parts: the functional reconstitution of the Arabidopsis thaZiana uncoupling protein 1 (AtPUMPI) and the expression and activity of the uncoupling protein under several abiotic stresses. AtPUMPI expression in Saccharomyces cerevisae and Escherichia coZi allowed us to confirm that this gene codes for a true uncoupling protein, able to divert energy of the proton electrochemical gradient. In a thermoneutral environment (28°C), AtPUMPI expressing cells grew slower than control cells, while under low temperatures (14 °C) growth rates were similar. When expressed in a prokaryotic system and reconstituted in lipid vesicles, AtPUMPI showed similar biochemical patterns to those already described for other uncoupling proteins. Ir transport was induced by severa! fatty acids and was dependent on the length and insaturations of the alkyl chain of the substrates. Pmine nucleotides were able to inhibit this proton transport, while changes in external pH alter AtPUMPl affinity for the nucleotides. Reconstitution experiments helped us to determine the Km and the Vmax for AtPUMPl. The values are within the physiologicallevels and very similar to those already obtained for other uncoupling proteins. We also studied the expression and activity of the uncoupling protein under situations of abiotic stresses, such as salinity, low temperature (chilling) and hyperoxygenated atmosphere. These situations are known to generate an oxidative stress. In maize coleoptiles, ZmPUMP mRNA expression increases under salt stress and does not change under chilling. However, protein expression and availability of substrates increased significantly on both situations, suggesting a post - transcriptional regulation for this gene. Moreover, mitochondria isolated ftom salt and cold stressed maize coleoptiles showed smaller ADP/O ratio, which returns to controllevels in the presence of BSA or GTP. In tomatoes submmited to a hyperoxygenated atmosphere, PUMP concentration and AOX activity patterns were similar to those already described for post-harvest ripening. However, preliminary experiments show increase in lipid peroxidation, resulting in a release of ftee fàtty acids, and consequent AOX inhibition and PUMP activation, even with a decrease in PUMP protein contento Under situations ofhigh intracellular Ca2+, we are able to observe also that uncoupling protein activators inhibit the mitochondrial generation of Reactive Oxygen Species (ROS) and that its inhibitors increased this generation. Considering all the above results, we ean conelude that AtPUMPI is a true uneoupling protein able to influenee eell growth through an uncoupling mechanism mediated by fatty aeids and inhibited by purine nucleotides. It is also possible to suggest that plant uneoupling proteins have a signifieant role under abiotic stress situations, decreasing mitoehondrial ROS generation and thus proteeting the tissue ttom the resulting oxidative stress Doutorado Ciências Biomédicas Doutor em Ciências Médicas