RESUMO
BACKGROUND: While nasopharyngeal (NP) swabs are considered the gold standard for severe acute respiratory coronavirus 2 (SARS-CoV-2) real-time reverse transcriptase-polymerase chain reaction (RT-PCR) detection, several studies have shown that saliva is an alternative specimen for COVID-19 diagnosis and screening. METHODS: To analyze the utility of saliva for the diagnosis of COVID-19 during the circulation of the Omicron variant, participants were enrolled in an ongoing cohort designed to assess the natural history of SARS-CoV-2 infection in adults and children. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Cohen's kappa coefficient were calculated to assess diagnostic performance. RESULTS: Overall, 818 samples were collected from 365 outpatients from January 3 to February 2, 2022. The median age was 32.8 years (range: 3-94 years). RT-PCR for SARS-CoV-2 was confirmed in 97/121 symptomatic patients (80.2%) and 62/244 (25.4%) asymptomatic patients. Substantial agreement between saliva and combined nasopharyngeal/oropharyngeal samples was observed with a Cohen's kappa value of 0.74 [95% confidence interval (CI): 0.67-0.81]. Sensitivity was 77% (95% CI: 70.9-82.2), specificity 95% (95% CI: 91.9-97), PPV 89.8% (95% CI: 83.1-94.4), NPV 87.9% (95% CI: 83.6-91.5), and accuracy 88.5% (95% CI: 85.0-91.4). Sensitivity was higher among samples collected from symptomatic children aged three years and older and adolescents [84% (95% CI: 70.5-92)] with a Cohen's kappa value of 0.63 (95% CI: 0.35-0.91). CONCLUSIONS: Saliva is a reliable fluid for detecting SARS-CoV-2, especially in symptomatic children and adolescents during the circulation of the Omicron variant.
Assuntos
COVID-19 , Pacientes Ambulatoriais , Adolescente , Adulto , Criança , Humanos , Saliva , Teste para COVID-19 , SARS-CoV-2/genética , COVID-19/diagnóstico , Nasofaringe , Manejo de EspécimesRESUMO
This study compares the effects of virus-cell interactions among SARS-CoV-2 variants of concern (VOCs) isolated in Brazil in 2021, hypothesizing a correlation between cellular alterations and mortality and between viral load and transmissibility. For this purpose, reference isolates of Alpha, Gamma, Zeta, and Delta variants were inoculated into monolayers of Vero-E6 cells. Viral RNA was quantified in cell supernatants by RTâPCR, and infected cells were analyzed by Transmission Electron Microscopy (TEM) for qualitative and quantitative evaluation of cellular changes 24, 48, and 72 hours postinfection (hpi). Ultrastructural analyses showed that all variants of SARS-CoV-2 altered the structure and function of mitochondria, nucleus, and rough endoplasmic reticulum of cells. Monolayers infected with the Delta variant showed the highest number of modified cells and the greatest statistically significant differences compared to those of other variants. Viral particles were observed in the cytosol and the cell membrane in 100 % of the cells at 48 hpi. Alpha showed the highest mean particle diameter (79 nm), and Gamma and Delta were the smallest (75 nm). Alpha and Gamma had the highest particle frequency per field at 48 hpi, while the same was observed for Zeta and Delta at 72 hpi and 24 hpi, respectively. The cycle threshold of viral RNA varied among the target protein, VOC, and time of infection. The findings presented here demonstrate that all four VOCs evaluated caused ultrastructural changes in Vero-E6 cells, which were more prominent when infection occured with the Delta variant.
Assuntos
COVID-19 , Citologia , Humanos , SARS-CoV-2 , RNA Viral/genéticaRESUMO
The SARS-CoV-2 P.1 variant, responsible for an outbreak in Manaus, Brazil, is distinguished by 12 amino acid differences in the S protein, potentially increasing its ACE-2 affinity and immune evasion capability. We investigated the innate immune response of this variant compared to the original B.1 strain, particularly concerning cytokine production. Blood samples from three severe COVID-19 patients were analyzed post-infection with both strains. Results showed no significant difference in cytokine production of mononuclear cells and neutrophils for either variant. While B.1 had higher cytopathogenicity, neither showed viral replication in mononuclear cells. Structural analyses of the S protein highlighted physicochemical variations, which might be linked to the differences in infectivity between the strains. Our studies point to the increased infectivity of P.1 could stem from altered immunogenicity and receptor-binding affinity.
RESUMO
The present study investigated a SARS-CoV-2 infection in placenta and fetal samples from an early pregnancy miscarriage in Midwest Brazil. The Gamma variant was isolated and fully sequenced from the placenta sample, but not from fetal samples. Our findings highlight potential adverse perinatal outcomes caused by SARS-CoV-2 Gamma infection during pregnancy.
RESUMO
Abstract This study compares the effects of virus-cell interactions among SARS-CoV-2 variants of concern (VOCs) isolated in Brazil in 2021, hypothesizing a correlation between cellular alterations and mortality and between viral load and transmissibility. For this purpose, reference isolates of Alpha, Gamma, Zeta, and Delta variants were inoculated into monolayers of Vero-E6 cells. Viral RNA was quantified in cell supernatants by RT‒PCR, and infected cells were analyzed by Transmission Electron Microscopy (TEM) for qualitative and quantitative evaluation of cellular changes 24, 48, and 72 hours postinfection (hpi). Ultrastructural analyses showed that all variants of SARS-CoV-2 altered the structure and function of mitochondria, nucleus, and rough endoplasmic reticulum of cells. Monolayers infected with the Delta variant showed the highest number of modified cells and the greatest statistically significant differences compared to those of other variants. Viral particles were observed in the cytosol and the cell membrane in 100 % of the cells at 48 hpi. Alpha showed the highest mean particle diameter (79 nm), and Gamma and Delta were the smallest (75 nm). Alpha and Gamma had the highest particle frequency per field at 48 hpi, while the same was observed for Zeta and Delta at 72 hpi and 24 hpi, respectively. The cycle threshold of viral RNA varied among the target protein, VOC, and time of infection. The findings presented here demonstrate that all four VOCs evaluated caused ultrastructural changes in Vero-E6 cells, which were more prominent when infection occured with the Delta variant.
RESUMO
The intracellular protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas' disease. We have previously characterized a T. cruzi virulence factor named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family. Single mutant parasite clones (Tc52(+/-)) exhibiting low virulence in vitro and in vivo were obtained by targeted Tc52 gene replacement. In this report, we have extended our study to analyze the immune response and the disease phenotype in Tc52(+/-)-infected BALB/c mice, during the acute and chronic phases of the disease. Significantly lower parasitemia were found in Tc52(+/-)-infected mice, as compared to wild-type parasite (WT)-infected ones. However, the expansion of all classes of lymphocytes and macrophages was similar for both clones. Furthermore, except for IgG2b levels which were higher in the case of WT-infected mice, all classes of Ig presented no significant difference for WT and Tc52(+/-)-infected animals. Interestingly, a lack of suppression of IL-2 production and of T-cell proliferation inhibition was observed in the case of spleen cells from Tc52(+/-)-infected mice. Finally, the pattern of inflammation process was different and characterized as diffused in the case of Tc52(+/-)-infected mice, or presenting numerous foci in the case of WT-infected mice. Localization of the Tc52 protein in tissue sections and infected heart cell primary cultures by immunofluorescence and immunogold labeling, respectively, revealed the presence of Tc52 at the amastigote surface and associated to aggregates within host cell vesicles. Taken together, these results reinforce the notion of Tc52 being a virulence factor playing a role in the phenotype of the immune response associated to the infection and on the course of the disease.
Assuntos
Doença de Chagas/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidade , Animais , Anticorpos Antiprotozoários/sangue , Apoptose , Células Cultivadas , Doença de Chagas/parasitologia , Doença de Chagas/patologia , Citocinas/biossíntese , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Marcação de Genes , Coração/parasitologia , Imunoglobulinas/sangue , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Músculo Esquelético/química , Músculo Esquelético/parasitologia , Músculo Esquelético/patologia , Miocárdio/química , Miocárdio/patologia , Proteínas de Protozoários/análise , Proteínas de Protozoários/imunologia , Baço/citologia , Baço/imunologia , Trypanosoma cruzi/imunologia , Trypanosoma cruzi/fisiologiaRESUMO
Macrophages are able to recognize, internalize and destroy a large number of pathogens, thus restricting the infection until adaptive immunity is initiated. In this work our aim was to analyze the surface charge of cells activated by carrageenan (CAR) and lipopolysaccharide (LPS) through light and electron microscopy approaches as well as the release of inflammatory mediators in vitro. The ultrastuctural analysis and the light microscopy data showed that in vivo administration of CAR represents a potent inflammatory stimulation for macrophages leading to a high degree of spreading, an increase in their size, in the number of the intracellular vacuoles and membrane projections as compared to the macrophages collected from untreated animals as well as mice submitted to LPS. Our data demonstrated that CAR stimulated-macrophages displayed a remarkable increase in nitric oxide production and PGE2 release as compared to the cells collected from non-stimulated and stimulated mice with LPS in vivo. On the other hand, non-stimulated macrophages as well as macrophages stimulated by LPS produce almost the same quantities of TNF-alpha, while in vivo stimulation by CAR leads to a 30-40% increase of cytokine release in vitro compared to the other groups. In conclusion, our morphological and biochemical data clearly showed that in vivo stimulation with CAR induces a potent inflammatory response in macrophages representing an interesting model to analyze inflammatory responses.
Assuntos
Carragenina/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Animais , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Movimento Celular/efeitos dos fármacos , Dinoprostona/metabolismo , Ferritinas/química , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Masculino , Camundongos , Óxido Nítrico/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/ultraestruturaRESUMO
The electrostatic surface charge and surface tension of mononuclear cells/monocytes obtained from young and adult marsupials (Didelphis marsupialis) were investigated by using cationized ferritin and colloidal iron hydroxyde, whole cell electrophoresis, and measuments of contact angles. Anionic sites were found distributed throughout the entire investigated cell surfaces. The results revealed that the anionic character of the cells is given by electrostatic charges corresponding to -18.8 mV (cells from young animals) and -29.3 mV (cells from adult animals). The surface electrostatic charge decreased from 10 to 65.2 per cent after treatment of the cells with each one of trypsin, neuraminidase and phospholipase C. The hydrophobic nature of the mononuclear cell surfaces studied by using the contact angle method revealed that both young and adult cells posses cell surfaces of high hidrofilicity since the angles formed with drops of saline water were 42.5º and 40.8º, respectively. Treatment of the cells, with trypsin or neuraminidase rendered their surfaces more hydrophobic, suggesting that sialic acid-containing glycoproteins are responsable for most of the hydrophilicity observed in the mononuclear cell surfaces from D. marsupialis.
Assuntos
Animais , Células , Monócitos , GambásRESUMO
Investigou-se algumas das propriedades básicas da superfície dos monócitos oriundos de marsupiais jovens e adultos (Didelphis marsupialis). A carga eletrostática de superfície e a tensäo superficial foram propriedades analisadas, pois elas têm importância crucial nos fenômenos relacionados a interaçöes celulares. Partículas catiônicas foram usadas para estudar a aniogenicidade da superfície dos monócitos e a topografia dos sítios iônicos da superfície. A nível ultra-estrutural mostrou-se que as superfícies analisadas säo negativas. Estes sítios aniônicos estäo distribuídos em toda superfície celular. Em algumas secçöes da superfície observou-se "clusters" de partículas catiônicas sugerindo que os sítios aniônicos foram capazes de se mover ao longo da membrana dos monócitos. A eletroforese celular dos monócitos foi usada para estimar o potencial eletrocinético dessas células. Os resultados revelaram que o caráter aniônico das células corresponde a -18,8 mV (células de animais jovens) e -29,3 MV (células de animais adultos) de potencial zeta. Para a identificaçäo de alguns dos componentes responsáveis pela negatividade dessas células realizaram-se ensaios de digestäo enzimática. Todas as superfícies estudadas tiveram a carga eletrostática diminuida após tratamento das células com tripsina, neuraminidase e fosfolipase C. A diminuiçäo da negatividade variou de 10 a 57,2 por cento para células previamente tratadas com as enzimas, fixadas e colocadas diretamente na cuba de eletroforese. A natureza hidrofóbica da superfície dos monócitos foi estudada usando-se o método do ângulo de contato. Tanto células de animais jovens quanto de animais adultos interagiram com salina altamente hidrofílica, sendo obtidos valores de 40,8§ e 42,5§, respectivamente, sendo que os valores obtidos foram alterados quando foram realizadas em células tratadas com as enzimas(AU)
Assuntos
Gambás , Monócitos/citologiaRESUMO
Patógenos e produtos microbianos vem sendo utilizados em estudos de macrófagos quanto à produçäo de citocinas e quimiocinas, essenciais para iniciar processos inflamatórios e a resposta imune contra organismos invasivos. A interaçäo inicial de parasitas intracelulares com macrófagos têm um importante papel na determinaçäo do equilíbrio parasita-hospedeiro. O Trypanosoma cruzi pode invadir muitos tipos celulares de hospedeiros vertebrados, ativando macrófagos, e assim reguando sua relaçäo com o hospedeiro. Neste trabalho analisamos a carga de superfície de células mononucleares de Didelphis marsupialis; a morfologia e funcionalidade de macrófagos de camundongos estimulados por carragenina lambda e LPS na sua interaçäo com T.cruzi, e o envolvimento da carragenina no processo de inflamaçäo no peritôneo destes animais. Observamos que a superfície das células de gambás adultos era mais negativa do que a de animais jovens. Em macrófagos de camundongos injetados com carragenina encontramos uma grande habilidade de ingestäo do corante lisossomal "lysotracker yellow". Devido a presença de numerosos vacúolos nestas células estudamos a origem destas organelas e a capacidade dos macrófagos em secretar a proteína inflamatória MRP-14. Observamos que esses vacúolos näo têm natureza lipídica e possuem uma grande reatividade para a proteína MRP-14, revelada por estudos de citoquímica ultra-estrutural. Nos estudos com camundongos estimulados com carragenina e infectados com T.cruzi, observamos que a parasitemia desses animais foi significativamente maior em comparaçäo com a de animais injetados apenas com salina. Macrófagos obtidos de animais estimulados in vivo por carragenina, quando infectados in vitro por T.cruzi apresentaram reduçäo da infecçäo. [...] Assim, concluímos que a carragenina lambda ativa macrófagos de camundongos que podem estar participando na modulaçäo da infecçäo por T. cruzi.(AU)
Assuntos
Animais , Camundongos , Macrófagos , Trypanosoma cruziRESUMO
Patógenos e produtos microbianos vem sendo utilizados em estudos de macrófagos quanto à produçäo de citocinas e quimiocinas, essenciais para iniciar processos inflamatórios e a resposta imune contra organismos invasivos. A interaçäo inicial de parasitas intracelulares com macrófagos têm um importante papel na determinaçäo do equilíbrio parasita-hospedeiro. O Trypanosoma cruzi pode invadir muitos tipos celulares de hospedeiros vertebrados, ativando macrófagos, e assim reguando sua relaçäo com o hospedeiro. Neste trabalho analisamos a carga de superfície de células mononucleares de Didelphis marsupialis; a morfologia e funcionalidade de macrófagos de camundongos estimulados por carragenina lambda e LPS na sua interaçäo com T.cruzi, e o envolvimento da carragenina no processo de inflamaçäo no peritôneo destes animais. Observamos que a superfície das células de gambás adultos era mais negativa do que a de animais jovens. Em macrófagos de camundongos injetados com carragenina encontramos uma grande habilidade de ingestäo do corante lisossomal "lysotracker yellow". Devido a presença de numerosos vacúolos nestas células estudamos a origem destas organelas e a capacidade dos macrófagos em secretar a proteína inflamatória MRP-14. Observamos que esses vacúolos näo têm natureza lipídica e possuem uma grande reatividade para a proteína MRP-14, revelada por estudos de citoquímica ultra-estrutural. Nos estudos com camundongos estimulados com carragenina e infectados com T.cruzi, observamos que a parasitemia desses animais foi significativamente maior em comparaçäo com a de animais injetados apenas com salina. Macrófagos obtidos de animais estimulados in vivo por carragenina, quando infectados in vitro por T.cruzi apresentaram reduçäo da infecçäo. [...] Assim, concluímos que a carragenina lambda ativa macrófagos de camundongos que podem estar participando na modulaçäo da infecçäo por T. cruzi.(AU)
Assuntos
Animais , Camundongos , Macrófagos , Trypanosoma cruziRESUMO
Investigou-se algumas das propriedades básicas da superfície dos monócitos oriundos de marsupiais jovens e adultos (Didelphis marsupialis). A carga eletrostática de superfície e a tensäo superficial foram propriedades analisadas, pois elas têm importância crucial nos fenômenos relacionados a interaçöes celulares. Partículas catiônicas foram usadas para estudar a aniogenicidade da superfície dos monócitos e a topografia dos sítios iônicos da superfície. A nível ultra-estrutural mostrou-se que as superfícies analisadas säo negativas. Estes sítios aniônicos estäo distribuídos em toda superfície celular. Em algumas secçöes da superfície observou-se "clusters" de partículas catiônicas sugerindo que os sítios aniônicos foram capazes de se mover ao longo da membrana dos monócitos. A eletroforese celular dos monócitos foi usada para estimar o potencial eletrocinético dessas células. Os resultados revelaram que o caráter aniônico das células corresponde a -18,8 mV (células de animais jovens) e -29,3 MV (células de animais adultos) de potencial zeta. Para a identificaçäo de alguns dos componentes responsáveis pela negatividade dessas células realizaram-se ensaios de digestäo enzimática. Todas as superfícies estudadas tiveram a carga eletrostática diminuida após tratamento das células com tripsina, neuraminidase e fosfolipase C. A diminuiçäo da negatividade variou de 10 a 57,2 por cento para células previamente tratadas com as enzimas, fixadas e colocadas diretamente na cuba de eletroforese. A natureza hidrofóbica da superfície dos monócitos foi estudada usando-se o método do ângulo de contato. Tanto células de animais jovens quanto de animais adultos interagiram com salina altamente hidrofílica, sendo obtidos valores de 40,8§ e 42,5§, respectivamente, sendo que os valores obtidos foram alterados quando foram realizadas em células tratadas com as enzimas(AU)