Epidemiological profile of Zika, Dengue and Chikungunya virus infections identified by medical and molecular evaluations in Rondonia, Brazil

Several arboviruses have emerged and/or re-emerged in North, Central and SouthAmerican countries. Viruses from some regions of Africa and Asia, such as the Zika and Chikungunya virus have been introduced in new continents causing major public health problems. The aim of this study was to investigate the presence of RNA from Zika, Dengue and Chikungunya viruses in symptomatic patients from Rondonia, where the epidemiological profile is still little known, by one-step real-time RT-PCR. The main clinical signs and symtoms were fever (51.2%), headache (78%), chills (6.1%), pruritus (12.2%), exanthema (20.1%), arthralgia (35.3%), myalgia (26.8%) and retro-orbital pain (19.5%). Serum from 164 symptomatic patients were collected and tested for RNA of Zika, Dengue types 1 to 4 and Chikungunya viruses, in addition to antibodies against Dengue NS1 antigen. Direct microscopy for Malaria was also performed. Only ZIKV RNA was detected in 4.3% of the patients, and in the remaining 95.7% of the patients RNA for Zika, Dengue and Chikungunya viruses were not detected. This finding is intriguing as the region has been endemic for Dengue for a long time and more recently for Chikungunya virus as well. The results indicated that medical and molecular parameters obtained were suitable to describe the first report of symptomatic Zika infections in this region. Furthermore, the low rate of detection, compared to clinical signs and symptoms as the solely diagnosis criteria, suggests that molecular assays for detection of viruses or other pathogens that cause similar symptoms should be used and the corresponding diseases could be included in the compulsory notification list

Comorbidities and causes of death in SARS-CoV-2-infected patients from the Amazon region / Comorbidades e causas de morte em pacientes infectados com SARS-CoV-2 na região amazônica

COVID-19 may be more severe in elder people and/or people presenting comorbidities, possibly leading to death. Therefore, we analyzed the profile of deaths caused by SARS-CoV-2 in part of the Amazon region, associating them to risk factors. 467 cases of death caused by COVID-19 from October 2020 to July 2021 were analyzed and correlated to age group, gender, comorbidities, and other risk factors, using the Chi-squared Test or Fisher's Exact Test as necessary. Deaths occurred in the age group between 17 and 98 years, with a predominance of men (57.4%) and higher concentration in the period between March and April 2021. Systemic arterial hypertension was the most prevalent disease, followed by smoking, cardiovascular disease, and diabetes mellitus. Smoker men and obese women (and/or with cardiovascular disease) presented higher chances to die, as well as obese people under 65 years and people over 65 years with cardiovascular disease, smokers, or hypertensive (p<0.05). The description of risk groups contributes for the adoption of strategies directed to the most vulnerable populations, as disease monitoring and an increase in vaccination rate, reducing the probability of overloading Brazil's Unified Health System

Genômica funcional da infecção de cardiomiócitos por Trypanosoma cruzi in vitro

Submitted by Alessandra Portugal ([email protected]) on 2013-09-17T14:37:08Z No. of bitstreams: 1 TESE RITA DE CASSIA PONTELLO RAMPAZZO.pdf: 4806860 bytes, checksum: 8062bd9dd8843e24004e4dfeb47291c3 (MD5)Made available in DSpace on 2013-09-17T14:37:08Z (GMT). No. of bitstreams: 1 TESE RITA DE CASSIA PONTELLO RAMPAZZO.pdf: 4806860 bytes, checksum: 8062bd9dd8843e24004e4dfeb47291c3 (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.A doença de Chagas, causada pelo protozoário Trypanosoma cruzi, acomete entre 7 e 8 milhões de pessoas especialmente na América Latina e Caribe, apresentando manifestações em órgãos associados ao sistema digestivo e cardíaco. A principal manifestação na fase crônica determinada é a cardiopatia chagásica e pode acometer entre 10 e 30% dos indivíduos infectados. Diante da ineficiência do tratamento, estudos com ênfase na biologia de interação parasita-hospedeiro têm sido desenvolvidos na busca de moléculas inovadoras que possam ser usadas para a prevenção, tratamento e/ou diagnóstico. A aplicação da genômic a funcional na interação T. cruzi-célula hospedeira tem como desafio desvendar a regulação de genes na célula alvo, buscando estratégias que enfoquem mecanismos de sobrevivência e replicação do parasita. A abordagem cinética utilizada neste trabalho (1, 2, 3, 4, 5, 6 e 24 horas de infecção) para analisar a expressão gênica global através da metodologia inovadora de RNA-Seq proporcionou um melhor entendimento dos eventos que culminam na ativação coordenada e temporal de moléculas durante a interação T. cruzi-cardiomiócito. Identificamos 572 genes modulados, sendo que 371 apresentavam expressão gênica induzida e 201 reprimidos. Nossos resultados revelam que o T. cruzi induz uma modulação gênica dinâmica com 18 genes alterados após uma hora de interação. No estágio inicial da infecção (1 a 4 horas), os genes modulados encontram-se relacionados com a estratégia de invasão do parasita e, principalmente, com a resposta de cardiomiócitos induzindo genes relacionados à resposta imune e atividade tripanocida. Neste contexto, evidenciamos a ativação de genes envolvidos com a resposta pró-inflamatória, estresse oxidativo e metabolismo de radicais livres indicativos da tentativa da célula cardíaca de conter a disseminação do parasita. Com o avanço da infecção, constatamos a modulação de genes integrados com o remodelamento do citoesqueleto e rigidez celular, provavelmente associado com a acomodação do parasita intracelular, e ainda repressão de genes relacionados com junções celulares e manutenção da arquitetura celular. Distúrbios na expressão de genes envolvidos em vias metabólicas mitocondriais apontam para uma deficiência energética e disfunção mitocondrial induzida pelo T. cruzi. Destacamos ainda alterações em genes associados à hipertrofia, principalmente relacionados à IL1 e IL6, receptor toll-like 2 e GSK3B, desde os tempos iniciais de infecção (1 a 4 horas). O remodelamento associado à matriz extracelular apresentou genes com padrão diminuído principalmente no ponto de 24 horas quando a taxa de infecção ultrapassa 75%, sendo as alterações relacionadas aos genes de colágeno e fibronectina. Genes associados à apoptose com perfil pró- e anti-apoptótico foram diferencialmente modulados nas diferentes fases da infecção, sugerindo que a indução ou repressão da apoptose pode estar diretamente relacionada a dispersão do parasita, controle da carga parasitária no hospedeiro e/ou escape da resposta imune Dessa forma, a identificação de genes importantes para adaptação do T. cruzi no microambiente da célula cardíaca aliada à modulação de genes envolvidos na resposta desta célula alvo contribuirá para aplicação de novas estratégias de controle deste parasita, possibilitando a seleção de potenciais alvos vacinais e/ou terapêuticos.Chagas` disease, caused by Trypanosoma cruzi, affects between 7 and 8 million people especially in Latin America and Caribbean, presenting manifestations in organs associated with the digestive system and heart. The main clinical manifestation on symptomatic chronic phase is chagasic cardiophaty and may affect about 10 to 30% of infected individuals. Given the ineffectiveness of the treatment, studies with emphasis on biology of host-pathogen interaction have been carried out to search innovative molecules that can be used to prevention, treatment and/or diagnosis. The application of functional genomics in the T. cruzi-host cell interaction has as its goal to display the regulation of genes in the target cell, seeking strategies that focus on mechanisms of survival and replication of the parasite. The kinetic approach used on this research (1, 2, 3, 4, 5, 6 and 24 hours of infection) to analyse the global gene expression by RNA-Seq, an innovative methodology, provide a better understanding of events that culminate in coordinated and temporal activation of molecules during T. cruzi-cardiomyocyte interaction. We identified 572 modulated genes, where 371 were induced and 201 repressed. Our results indicate that T. cruzi induces a dynamic gene modulation with 18 genes altered after one hour of interaction. On early stage of infection (1 to 4 hours), the modulated genes are related to immune response and trypanocydal activity. In this context, we observed the activation of genes involved in pro-inflammatory response, oxidative stress and free radical metabolism indicating that the cardiac cell attempts to control the dissemination of the parasite. With the progression of the infection, it was evident the modulation of genes involved in cytoskeleton remodeling and cell stiffness, probably associated with intracellular parasite accommodation and repression of cell junctions genes as well as cell architecture maintenance genes. Alterations in expression of genes involved in metabolic pathways point to a mitochondrial dysfunction and energy deficiency induced by T. cruzi. We also point out changes in genes associated with hypertrophy, mainly related to IL1, IL6, toll like receptor 2 and GSK3B from early times of infection (1 to 4 hours). Remodeling associated to extracellular matrix showed genes with decreased expression mainly at 24 hours when the infection exceeds 75%, specially collagen- and fibronectin-related genes. In addition, genes associated to apoptosis with pro- and anti- apoptotic activity were differentially modulated in different stages of infection, suggesting that induction or repression of apoptosis may be directly related to parasite spread, the parasite load control in the host and/or escape of immune response. Thus, the identification of genes important to adaptation of T. cruzi on the microenviroment of the cardiac cell combined with modulation of genes involved in the response of the target cell will contribute to implementation of new strategies to control this parasite, allowing the selection of potential vaccine and/or therapeutic targets

Current Nucleic Acid extraction methods and their implications to point-of-care diagnostics

Submitted by Manoel Barata ([email protected]) on 2017-08-29T19:21:24Z No. of bitstreams: 1 NasirCurrentNuc.pdf: 1319786 bytes, checksum: cc1a526713bc8ca6c156102cd76bed4e (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2017-09-12T12:26:13Z (GMT) No. of bitstreams: 1 NasirCurrentNuc.pdf: 1319786 bytes, checksum: cc1a526713bc8ca6c156102cd76bed4e (MD5)Made available in DSpace on 2017-09-12T12:26:13Z (GMT). No. of bitstreams: 1 NasirCurrentNuc.pdf: 1319786 bytes, checksum: cc1a526713bc8ca6c156102cd76bed4e (MD5) Previous issue date: 2017Universidade Federal do Paraná. Departamento de Engenharia de Bioprocessos e Biotecnologia. Curitiba, PR, Brasil / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Universidade Federal do Paraná. Departamento de Engenharia de Bioprocessos e Biotecnologia. Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Nucleic acid extraction (NAE) plays a vital role in molecular biology as the primary step for many downstream applications. Many modifications have been introduced to the original 1869 method. Modern processes are categorized into chemical or mechanical, each with peculiarities that influence their use, especially in point-of-care diagnostics (POC-Dx). POC-Dx is a new approach aiming to replace sophisticated analytical machinery with microanalytical systems, able to be used near the patient, at the point of care or point of need. Although notable efforts have been made, a simple and effective extraction method is still a major challenge for widespread use of POC-Dx. In this review, we dissected the working principle of each of the most common NAE methods, overviewing their advantages and disadvantages, as well their potential for integration in POC-Dx systems. At present, it seems difficult, if not impossible, to establish a procedure which can be universally applied to POC-Dx. We also discuss the effects of the NAE chemicals upon the main plastic polymers used to mass produce POC-Dx systems. We end our review discussing the limitations and challenges that should guide the quest for an efficient extraction method that can be integrated in a POC-Dx system

External control viral-like particle construction for detection of emergent Arboviruses by real-time reverse-transcription PCR

Submitted by Manoel Barata ([email protected]) on 2019-12-04T17:17:15Z No. of bitstreams: 1 BMRI2019-25604ok.pdf: 1048630 bytes, checksum: a8f1a3ae8fad241e2707cfd63c41f3c7 (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2020-02-12T14:23:15Z (GMT) No. of bitstreams: 1 BMRI2019-25604ok.pdf: 1048630 bytes, checksum: a8f1a3ae8fad241e2707cfd63c41f3c7 (MD5)Made available in DSpace on 2020-02-12T14:23:15Z (GMT). No. of bitstreams: 1 BMRI2019-25604ok.pdf: 1048630 bytes, checksum: a8f1a3ae8fad241e2707cfd63c41f3c7 (MD5) Previous issue date: 2019Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil / Universidade Federal do Paraná. Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Escritório Rondônia. Porto Velho, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil / Universidade Federal do Paraná. Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Curitiba, PR, Brasil / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Arboviruses have been emerging and reemerging worldwide, predominantly in tropical and subtropical areas. As many arbovirus infections, including dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV), have similar signs and symptoms, clinical diagnosis of arbovirus infections is challenging. Therefore, reliable laboratory tests are necessary to improve the clinical management of patients with suspected arbovirus infections. Real-time reverse-transcription PCR (RT-qPCR) is among the more effective methods to distinguish these viruses. The aim of this study was to construct a unique positive external control derived from a unique plasmid using genetic engineering for specific use in RT-qPCR assays to detect Zika, dengue (1-4), and chikungunya. An external control derived from the MS2 bacteriophage was constructed using sequences from arbovirus and human genomes. Laboratories were asked to test the control in the ZDC Biomol kit, a RT-qPCR kit which is able to detect Zika, dengue serotypes 1-4, chikungunya, and an internal human control. RNA extracted from the external control was able to be amplified and detected in RT-qPCR assays for each virus detected by using the ZDC Biomol kit. The external control, samples from viral culture, and infected patient samples display similar amplification using this assay. The pET47b(+)MS2-ZDC vector is a viable expression system for the production of external control viral-like particles (MS2-ZDC). The RNA from the recombinant particles can be easily extracted and can function as a tool to validate all steps of process from the extraction to the amplification of all targets in specific reaction. Thus, the MS2-ZDC particles are laboratory-safe in order to avoid risk for operators, and the phages are effective as positive control for use in the ZDC Biomol kit amplifying all kit targets making them effective for commercial profile

Yeasts and filamentous fungi in bottled mineral water and tap water from municipal supplies

The main objective of this study was to analyse the occurrence of yeasts and filamentous fungi in drinking water as well as to investigate their correlation with the indicator bacteria of faecal pollution. Yeasts were detected in 36.6% and 11.6% of the bottled mineral on water dispensers and tap water samples from municipal system, respectively. Twenty-one (35.0%) of bottled mineral water and two (3.3%) of tap water samples were positive for filamentous fungi. For bottled mineral water 12 (20.0%) of 60 samples were positive for total coliform, compared with 3(5.0%)out of 60 samples from tap water. The mineral water from dispensers was more contaminated than tap water. Strains belonging to the genera Candida identified to the species level were C. parapsilosis, C. glabrata and C. albicans. Thus, bottled mineral water from water dispensers and tap water could be considered a possible transmission route for filamentous fungi and yeasts, and could constitute a potential health hazard, mainly to immunocompromised indivuals.O principal objetivo do presente estudo foi avaliar a prevalência de leveduras e fungos filamentosos em água potável, bem como investigar suas correlações com bactérias indicadoras de contaminação fecal. Leveduras foram detectadas em 36,6% e 11,6% das amostras de água mineral de garrafão em dispensadores de água e água de torneira do sistema municipal, respectivamente. Vinte e uma (35,5%) das amostras de água mineral de garrafão e duas (3,3%) das amostras de água de torneira foram positivas para fungos filamentosos. Para água mineral de garrafão, 12 (20.0%) das 60 amostras foram positivas para coliforme total, comparado com 3 (5.0%) das 60 amostras de água de torneira. A água coletada de garrafões de água mineral dos dispensadores foi marcadamente mais contaminada que as amostras de água de torneira. Candida spp identificadas ao nível de espécie foram C. parapsilosis, C. glabrata e C. albicans. Como está sendo reportado, água mineral de garrafão em dispensador e água de torneira pode ser considerada como possíveis vias de transmissão de fungos filamentosos e leveduras, e podem constituir um potencial risco para a saúde, principalmente de pessoas imunocomprometidas

Profiling the Trypanosoma cruzi phosphoproteome

Submitted by Manoel Barata ([email protected]) on 2019-12-03T17:47:47Z No. of bitstreams: 1 journal.pone.002538ok.PDF: 411332 bytes, checksum: 1c64dda8f58ad34d9bd06e475d53ef4a (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2020-02-19T14:39:34Z (GMT) No. of bitstreams: 1 journal.pone.002538ok.PDF: 411332 bytes, checksum: 1c64dda8f58ad34d9bd06e475d53ef4a (MD5)Made available in DSpace on 2020-02-19T14:39:34Z (GMT). No. of bitstreams: 1 journal.pone.002538ok.PDF: 411332 bytes, checksum: 1c64dda8f58ad34d9bd06e475d53ef4a (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Department of Proteomics and Signal Transduction. Max Planck Institute of Biochemistry. Martinsried, Germany.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Department of Proteomics and Signal Transduction. Max Planck Institute of Biochemistry. Martinsried, Germany.Department of Proteomics and Signal Transduction. Max Planck Institute of Biochemistry. Martinsried, Germany.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Protein phosphorylation is a reversible post-translational modification essential for the regulation of several signal transduction pathways and biological processes in the living cell. Therefore, the identification of protein phosphorylation sites is crucial to understand cell signaling control at the molecular level. Based on mass spectrometry, recent studies have reported the large-scale mapping of phosphorylation sites in various eukaryotes and prokaryotes. However, little is known about the impact of phosphorylation in protozoan parasites. To in depth characterize the phosphoproteome of Trypanosoma cruzi, a parasite of the Kinetoplastida class, protein samples from cells at different phases of the metacyclogenesis--differentiation process of the parasites from non-infective epimastigotes to infective metacyclic trypomastigotes--were enriched for phosphopeptides using TiO(2) chromatography and analyzed on an LTQ-Orbitrap mass spectrometer. In total, 1,671 proteins were identified, including 753 phosphoproteins, containing a total of 2,572 phosphorylation sites. The distribution of phosphorylated residues was 2,162 (84.1%) on serine, 384 (14.9%) on threonine and 26 (1.0%) on tyrosine. Here, we also report several consensus phosphorylation sequence motifs and as some of these conserved groups have enriched biological functions, we can infer the regulation by protein kinases of this functions. To our knowledge, our phosphoproteome is the most comprehensive dataset identified until now for Kinetoplastida species. Here we also were able to extract biological information and infer groups of sites phosphorylated by the same protein kinase. To make our data accessible to the scientific community, we uploaded our study to the data repositories PHOSIDA, Proteome Commons and TriTrypDB enabling researchers to access information about the phosphorylation sites identified here

Knockout of the gene encoding the kinetoplast-associated protein 3 (KAP3) in Trypanosoma cruzi: effect on kinetoplast organization, cell proliferation and differentiation

9 p. : il.Kinetoplast DNA (kDNA) of trypanosomatid protozoa consists of an unusual arrangement of two types of circular molecules catenated into a single network: (1) a few maxicircles that encode various mitochondrial enzyme subunits and rRNA in a cryptic pattern and (2) thousands of minicircles that encode guide RNAs (gRNAs). kDNA is associated with proteins, known as kinetoplast-associated proteins (KAPs), which condense the kDNA network. However, little is known about the KAPs of Trypanosoma cruzi, a parasite that displays different kDNA condensation patterns during its complex morphogenetic development. We cloned the T. cruzi gene encoding TcKAP3 (kinetoplast-associated protein 3). TcKAP3 is a single-copy gene that is transcribed into a 1.8-kb mRNA molecule and expressed in all stages of the parasite. Mouse antiserum raised against recombinant TcKPA3 recognized a 21.8 kDa protein, which was found, by indirect immunofluorescence and immunoelectron microscopy, to be associated with the T. cruzi kinetoplast. Several features of TcKAP3, such as its small size, basic nature and similarity with KAP3 from the insect trypanosomatid Crithidia fasciculata, are consistent with a role in DNA charge neutralization and condensation. This suggests that this protein is involved in organizing the kDNA network. Gene deletion was used to investigate TcKAP3 function. Here we investigated the T. cruzi KAP3 null mutant, analyzing its fitness during proliferation, differentiation and infectivity