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Background & objectives: The oropharyngeal (OP) and nasopharyngeal (NP) swab samples are the most recommended clinical specimens for detecting SARS-CoV-2 in an individual through the quantitative real-time reverse-transcriptase-polymerase chain reaction (rRT-PCR) method. The primary objective of this study was to compare the performance of NP and OP swabs for the diagnosis of COVID-19 among 2250 concomitant samples (1125 NP + 1125 OP) using rRT-PCR test. Methods: This study was conducted at a tertiary care hospital in southern India. The study compared the specificity and efficacy of the two samples (NP & OP swabs) in 1125 individuals suspected having COVID-19 infection. The rRT-PCR values from all the samples were compared based on gender, age group and viral load. The differences between unmatched proportion and matched proportion were analysed. Agreement between the two methods was assessed using Kappa statistic. Absolute sensitivity, specificity, positive and negative predictive values (PPV and NPV) for OP and NP swabs were analysed. Results: The study identified a fair degree of agreement between OP and NP swabs in diagnosis of COVID-19 (kappa = 0.275, P <0.001). There was also a fair degree of agreement between NP and OP swabs irrespective of gender, age or duration of symptoms. NP swabs had better sensitivity and NPV as compared to OP swabs, however, specificity and PPV were 100 per cent for both. Interpretation & conclusions: The present study showed that both OP and NP swabs had similar sensitivity and specificity for predicting the presence of SARS-CoV-2.
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COVID-19 , Humanos , SARS-CoV-2 , Nasofaringe , Orofaringe , Reação em Cadeia da Polimerase em Tempo RealRESUMO
This corrects the article DOI: 10.1038/ncomms15865.
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Optimal regulation of the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is essential for controlling bacterial infections and inflammatory disorders. Chronic NOD2 stimulation induces non-responsiveness to restimulation, termed NOD2-induced tolerance. Although the levels of the NOD2 adaptor, RIP2, are reported to regulate both acute and chronic NOD2 signalling, how RIP2 levels are modulated is unclear. Here we show that ZNRF4 induces K48-linked ubiquitination of RIP2 and promotes RIP2 degradation. A fraction of RIP2 localizes to the endoplasmic reticulum (ER), where it interacts with ZNRF4 under either 55 unstimulated and muramyl dipeptide-stimulated conditions. Znrf4 knockdown monocytes have sustained nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, and Znrf4 knockdown mice have reduced NOD2-induced tolerance and more effective control of Listeria monocytogenes infection. Our results thus demonstrate E3-ubiquitin ligase ZNRF4-mediated RIP2 degradation as a negative regulatory mechanism of NOD2-induced NF-κB, cytokine and anti-bacterial responses in vitro and in vivo, and identify a ZNRF4-RIP2 axis of fine-tuning NOD2 signalling to promote protective host immunity.
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Acetilmuramil-Alanil-Isoglutamina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Tolerância Imunológica , Proteína Adaptadora de Sinalização NOD2/metabolismo , Acetilmuramil-Alanil-Isoglutamina/imunologia , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Células HEK293 , Humanos , Tolerância Imunológica/efeitos dos fármacos , Listeria monocytogenes/patogenicidade , Listeriose/imunologia , Listeriose/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Monócitos/metabolismo , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Transdução de Sinais/fisiologia , Ubiquitinação/efeitos dos fármacosRESUMO
Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin-mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI.
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Infecções por Escherichia coli/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Infecções Urinárias/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Acetilcisteína , Animais , Linhagem Celular , Escherichia coli/metabolismo , Feminino , Imunofluorescência , Humanos , Ferro/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Reação em Cadeia da Polimerase , Transporte Proteico/fisiologia , Transfecção , Bexiga Urinária/microbiologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/metabolismoRESUMO
The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon-ß production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7) were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by ß-phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications.