Label-free aptasensor for p24-HIV protein detection based on graphene quantum dots as an electrochemical signal amplifier

Human immunodeficiency virus (HIV) is still considered a pandemic, and the detection of p24-HIV protein has an important role in the early diagnosis of HIV in adults and newborns. The accessibility of these trials depends on the price and execution difficulty of the method, which can be reduced using electrochemical methods by using enzymeless approaches, disposable and accurate devices. In this work, graphene quantum dots were acquired by a simple synthesis and employed as an electrochemical signal amplifier and support for the aptamer immobilization through a feasible and stable modification of disposable screen-printed electrodes. The device has been easily assembled and used to detect p24-HIV protein without the interference of similar proteins or sample matrix. Using the best set of experimental conditions, a linear correlation between analytical signal and log of p24-HIV concentration from 0.93 ng mL−1 to 93 μg mL−1 and a limit of detection of 51.7 pg mL−1 were observed. The developed device was applied to p24 determination in spiked human serum and provided distinct levels of signal for positive and negative samples, successfully identifying real samples with the target protein. This sensor is a step towards the development of point-of-care devices and the popularization of electrochemical methods for trials and diagnostics of relevant diseases

Evaluation of eleven immunochromatographic assays for SARS-CoV-2 detection: investigating the dengue cross-reaction

COVID-19 disease is spread worldwide and diagnostic techniques have been studied in order to contain the pandemic. Immunochromatographic (IC) assays are feasible and a low-cost alternative especially in low and middle-income countries, which lack structure to perform certain diagnostic techniques. Here we evaluate the sensitivity and specificity of eleven different IC tests in 145 serum samples from confirmed cases of COVID-19 using RT-PCR and 100 negative serum samples from blood donors collected in February 2019. We also evaluated the cross-reactivity with dengue using 20 serum samples from patients with confirmed diagnosis for dengue collected in early 2019 through four different tests. We found high sensitivity (92%), specificity (100%) and an almost perfect agreement (Kappa 0.92) of IC assay, especially when we evaluated IgG and IgM combined after 10 days from the onset of symptoms with RT-PCR. However, we detected cross-reactivity between dengue and COVID-19 mainly with IgM antibodies (5 to 20% of cross-reaction) and demonstrated the need for better studies about diagnostic techniques for these diseases

Predicting the Proteins of Angomonas deanei, Strigomonas culicis and Their Respective Endosymbionts Reveals New Aspects of the Trypanosomatidae Family

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. in an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. the monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. the monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. the assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)ERC AdG SISYPHEUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Lab Ultraestrutura Celular Hertha Meyer, BR-21941 Rio de Janeiro, BrazilUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Lab Metab Macromol Firmino Torres de Castro, BR-21941 Rio de Janeiro, BrazilLab Bioinformat, Lab Nacl Computacao Cient, Rio de Janeiro, BrazilINRIA Grenoble Rhone Alpes, BAMBOO Team, Villeurbanne, FranceUniv Lyon 1, CNRS, UMR5558, Lab Biometrie & Biol Evolut, F-69622 Villeurbanne, FranceUniv Estadual Campinas, Inst Biol, Dept Genet Evolucao & Bioagentes, São Paulo, BrazilUniv São Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Dept Ciencias Farmaceut, São Paulo, BrazilLab Nacl Ciencia & Tecnol Bioetano, São Paulo, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Bioquim & Imunol, Belo Horizonte, MG, BrazilUniv Fed Goias, Inst Ciencias Biol, Mol Biol Lab, Goiania, Go, BrazilFundacao Oswaldo Cruz, Inst Carlos Chagas, Lab Biol Mol Tripanossomatideos, Curitiba, Parana, BrazilFundacao Oswaldo Cruz, Inst Carlos Chagas, Lab Genom Func, Curitiba, Parana, BrazilUniv Estadual Campinas, Ctr Pluridisciplinar Pesquisas Quim Biol & Agr, São Paulo, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Parasitol, Belo Horizonte, MG, BrazilUniv Fed Santa Catarina, Dept Microbiol Imunol & Parasitol, Ctr Ciencias Biol, Lab Protozool & Bioinformat, Florianopolis, SC, BrazilUniv Fed Vicosa, Dept Bioquim & Biol Mol, Ctr Ciencias Biol & Saude, Vicosa, MG, BrazilInst Butantan, Lab Especial Ciclo Celular, São Paulo, BrazilUniv São Paulo, Dept Biol, Fac Filosofia Ciencias & Letras Ribeirao Preto, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWeb of Scienc

Análise pós-genômica de via de transduçao de sinal mediado por AMPc durante o ciclo de vida do Trypanosoma cruzi

Orientador : Marco Aurelio KriegerDissertaçao (mestrado) - Universidade Federal do Paraná, Setor de Ciencias Biológicas, Programa de Pós-Graduaçao em Biologia Celular Molecular. Defesa: Curitiba, 25/06/2004Inclui bibliografia e anexoO processo de diferenciação celular de epimastigotas para tripomastigotas metacíclicos em Trypanosoma cruzi, bem como alguns genes e fatores envolvidos neste processo, incluindo a importância do AMPc como segundo mensageiro na indução da metaciclogênese, vêm sendo caracterizados por nosso grupo. Para melhor entender a via de transdução de sinal do AMPc, foram realizadas buscas em bancos de seqüência, por genes que codificariam para diferentes proteínas relacionadas com esta via de sinalização (adenilato ciclases, fosfodiesterases e genes codificando as subunidades da holoenzima PKA), permitindo selecionar diferentes membros destas famílias gênicas e objetivando criar um cenário para esta via de transdução de sinal. Para quantificar os transcritos destes genes selecionados ao longo do ciclo de vida do T. cruzi, oligonucleotídeos iniciadores foram sintetizados, e a quantificação foi feita por PCR em tempo real. O conjunto de dados sugere que esses genes sofrem uma regulação complexa e coordenada de sua expressãoThe cellular differentiation process from epimastigote to metacyclic tripomastigote in Trypanosoma cruzi , as well as some genes and factors involved in this process, have been characterized by our group including the role of cAMP as an important secondary messenger in inducing metacyclogenesis. We have searched in the sequence data banks for genes that encode different proteins related to this transduction pathway (Adenylate Cyclases, Phosphodiesterases and the genes for the holoenzyme cAMP dependent Protein Kinase), allowing us to selected different members of these genes family. In order to quantify their gene expression during the T. cruzi life cycle we have developed primers and probes for Real time RT-PCR. Our data strongly suggest that genes related to this pathway have a complex and coordinate gene expression regulation

Análise pós-genômica de via de transduçao de sinal mediado por AMPc durante o ciclo de vida do Trypanosoma cruzi

Orientador : Marco Aurelio KriegerDissertaçao (mestrado) - Universidade Federal do Paraná, Setor de Ciencias Biológicas, Programa de Pós-Graduaçao em Biologia Celular Molecular. Defesa: Curitiba, 25/06/2004Inclui bibliografia e anexoO processo de diferenciação celular de epimastigotas para tripomastigotas metacíclicos em Trypanosoma cruzi, bem como alguns genes e fatores envolvidos neste processo, incluindo a importância do AMPc como segundo mensageiro na indução da metaciclogênese, vêm sendo caracterizados por nosso grupo. Para melhor entender a via de transdução de sinal do AMPc, foram realizadas buscas em bancos de seqüência, por genes que codificariam para diferentes proteínas relacionadas com esta via de sinalização (adenilato ciclases, fosfodiesterases e genes codificando as subunidades da holoenzima PKA), permitindo selecionar diferentes membros destas famílias gênicas e objetivando criar um cenário para esta via de transdução de sinal. Para quantificar os transcritos destes genes selecionados ao longo do ciclo de vida do T. cruzi, oligonucleotídeos iniciadores foram sintetizados, e a quantificação foi feita por PCR em tempo real. O conjunto de dados sugere que esses genes sofrem uma regulação complexa e coordenada de sua expressãoThe cellular differentiation process from epimastigote to metacyclic tripomastigote in Trypanosoma cruzi , as well as some genes and factors involved in this process, have been characterized by our group including the role of cAMP as an important secondary messenger in inducing metacyclogenesis. We have searched in the sequence data banks for genes that encode different proteins related to this transduction pathway (Adenylate Cyclases, Phosphodiesterases and the genes for the holoenzyme cAMP dependent Protein Kinase), allowing us to selected different members of these genes family. In order to quantify their gene expression during the T. cruzi life cycle we have developed primers and probes for Real time RT-PCR. Our data strongly suggest that genes related to this pathway have a complex and coordinate gene expression regulation

Identification of Secreted Proteins Involved in Nonspecific dsRNA-Mediated Lutzomyia longipalpis LL5 Cell Antiviral Response

Submitted by Sandra Infurna ([email protected]) on 2019-02-14T13:43:03Z No. of bitstreams: 1 yaramariat_cseko_etal_IOC_2018.pdf: 2048797 bytes, checksum: b315ee0381efe6114c33e4d8e230ef8b (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-02-14T13:52:25Z (GMT) No. of bitstreams: 1 yaramariat_cseko_etal_IOC_2018.pdf: 2048797 bytes, checksum: b315ee0381efe6114c33e4d8e230ef8b (MD5)Made available in DSpace on 2019-02-14T13:52:25Z (GMT). No. of bitstreams: 1 yaramariat_cseko_etal_IOC_2018.pdf: 2048797 bytes, checksum: b315ee0381efe6114c33e4d8e230ef8b (MD5) Previous issue date: 2018Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Plataforma Espectrometria de Massas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Plataforma Espectrometria de Massas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ. Brasil.Hematophagous insects transmit infectious diseases. Sand flies are vectors of leishmaniasis, but can also transmit viruses. We have been studying immune responses of Lutzomyia longipalpis, the main vector of visceral leishmaniasis in the Americas. We identified a non-specific antiviral response in L. longipalpis LL5 embryonic cells when treated with non-specific double-stranded RNAs (dsRNAs). This response is reminiscent of interferon response in mammals. We are investigating putative effectors for this antiviral response. Secreted molecules have been implicated in immune responses, including interferon-related responses. We conducted a mass spectrometry analysis of conditioned medium from LL5 cells 24 and 48 h after dsRNA or mock treatment. We identified 304 proteins. At 24 h, 19 proteins had an abundance equal or greater than 2-fold change, while the levels of 17 proteins were reduced when compared to control cells. At the 48 h time point, these numbers were 33 and 71, respectively. The two most abundant secreted peptides at 24 h in the dsRNA-transfected group were phospholipid scramblase, an interferon-inducible protein that mediates antiviral activity, and forskolin-binding protein (FKBP), a member of the immunophilin family, which mediates the effect of immunosuppressive drugs. The transcription profile of most candidates did not follow the pattern of secreted protein abundance

LC-ESI-MS/MS NA IDENTIFICAÇÃO DE AGENTES ETIOLÓGICOS DA SEPSE

Introdução/Objetivos: Sepse é a disfunção múltipla de órgãos causada pela resposta inflamatória irregular do corpo a uma infecção, incluída como prioridade em Saúde Pública pela Organização Mundial de Saúde. É a maior causa de mortes entre pacientes admitidos UTIs, o que é associado à falta de diagnóstico/tratamento eficiente em tempo adequado. O diagnóstico por hemocultura considerado padrão ouro na diagnose, demanda de 3 a 7 dias para o resultado final e baixa sensibilidade. Avanços têm sido realizados na identificação de patógenos a partir de hemocultura positiva, como automação dos testes fenotípicos e bioquímicos, testes moleculares e espectrometria de massas (MS) pela técnica MALDI-TOF, porém a dependência do cultivo prévio ocasiona importantes limitações, com aproximadamente 70% de resultados falso negativos e longo tempo necessário para o crescimento (1 a 5 dias ou mais). Neste estudo desenvolvemos uma prova de conceito para metodologia baseada em LC-MS/MS com o objetivo de monitorar íons de alta intensidade específicos para micro-organismos relacionados à sepse, diretamente de amostras de sangue total, sem a necessidade de cultivo microbiológico. Metodologia: O método que desenvolvemos tem como etapas metodológicas partindo de amostra de sangue total: lise diferencial de pH básico e lise celular ácida, extração/digestão rápida das proteínas do microrganismo e o uso da LC/ESI-MS/MS na análise/identificação dos peptídeos únicos discriminatórios de cada patógeno estudado. Resultados: Demonstramos a eficácia de nossa metodologia ao diagnosticar amostras infectadas com um ou mais dos seguintes patógenos: S. aureus, P. aeruginosa e C. albicans. Nosso método selecionou peptídeos discriminantes a partir dos dados gerados por LC-MS/MS que forneceram identificações corretas para todos os microrganismos mencionados acima com sensibilidade de 87,5% em sete horas e sem necessidade de enriquecimento em microcultura. Conclusão: Apresentamos um procedimento simples e rápido para a pré-seleção de um painel de peptídeos a ser usado para diagnóstico. A vantagem do nosso método é que podemos diagnosticar patógenos diretamente do sangue total, ao invés de passar pelo processo de cultura, configurando uma alternativa diagnóstica para sangue infecção. Prevemos também que nosso método será útil na identificação de fungos filamentosos e no diagnóstico de resistência antimicrobiana, em última análise, contribuindo para dados epidemiológicos

Evaluating the diagnostic accuracy of TpN17 and TmpA recombinant proteins in syphilis detection: a phase II study

Syphilis is a sexually transmitted infection (STI) caused by the spiral bacterium Treponema pallidum. Diagnosis is based on epidemiology, clinical and serology, but serodiagnosis is challenging because distinct clinical forms of the infection may influence serological performance. Several recombinant Treponema pallidum-proteins have already been tested for syphilis diagnosis and they are critical to achieve high accuracy in serological testing. A total of 647 samples were included in the study: 180 T. pallidum-positive samples, 191 T. pallidum-negative samples and 276 sera from individuals infected with unrelated diseases. The diagnostic potential was validated by analysis of ROC curves. For the indirect ELISA, TpN17 (100%) and TmpA (99%) showed excellent AUC values. Sensitivity values were 97.2% for TpN17 and 90.6% for TmpA, while specificity was 100% for both molecules. According to the clinical phase, TmpA ranged from 84% to 97%, with the highest value for secondary syphilis. TpN17 was 100% sensitive for the primary and secondary stages and 93.2% for recent latent syphilis. All clinical phases achieved 100% specificity. Accuracy values showed that TmpA (> 95%) and TpN17 (> 98%) presented high diagnostic accuracy for all clinical stages of syphilis. Cross-reactivity was only observed in one sample positive for Chagas disease (1.5%), when TpN17 was evaluated. On the other hand, TmpA showed reactivity for two samples positive for Chagas disease (3.1%), one sample positive for HBV (1.25%), two samples positive for HIV (9.5%) and one sample positive for HTLV (1.6%). The TmpA antigen’s performance was evaluated in multiple studies for syphilis diagnosis, corroborating our findings. However, TpN17 sensitivity values have ranged in other studies. According to clinical stages of the infection, our findings obtained close performance values

Validation of the NAT Chagas IVD Kit for the Detection and Quantification of <i>Trypanosoma cruzi</i> in Blood Samples of Patients with Chagas Disease

In the absence of validated biomarkers to control the cure of Chagas disease, PCR-based diagnosis is being used as the main tool for an early indication of therapeutic failure. However, since it is considered a technique of complex reproducibility, mainly due to difficulties in establishing accurate controls to guarantee the quality of the reaction, the use of PCR for Chagas disease diagnosis is restricted to specialized centers. In an effort to disseminate the molecular diagnosis of Chagas disease and its applications, new diagnostic kits based on qPCR have been made available in the market in recent years. Here, we show the results of the validation of the NAT Chagas kit (Nucleic Acid Test for Chagas Disease) for the detection and quantification of T. cruzi in blood samples of patients suspected of Chagas disease infection. The kit, composed of a TaqMan duplex reaction targeting the T. cruzi satellite nuclear DNA and an exogenous internal amplification control, presented a reportable range from 104 to 0.5 parasite equivalents/mL and a limit of detection (LOD) of 0.16 parasite equivalents/mL of blood. In addition, the NAT Chagas kit detected T. cruzi belonging to all six discrete typing units (DTUs—TcI to TcVI), similarly to the in-house real-time PCR performed with commercial reagents, which has been selected as the best performance assay in the international consensus for the validation of qPCR for Chagas disease. In the clinical validation presented here, the kit showed 100% sensitivity and 100% specificity when compared to the consensus in-house real-time PCR assay. Thus, the NAT Chagas kit, which is produced entirely in Brazil under the international standards of good manufacturing practices (GMP), appears as an excellent alternative to enable the molecular diagnosis of Chagas disease in public and private diagnostic centers, as well as to facilitate the monitoring of patients under etiological treatment participating in clinical trials