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Here we report an ultrafast quadruplex RT-qPCR assay with robust diagnostic ability to detect and distinguish pan-SARS-CoVs and influenza A/B viruses within 35 min. This quadruplex RT-qPCR assay comprised of one novel RNA-based internal control targeting human ß2-microglobulin (B2M) for process accuracy and three newly-designed primers-probe sets targeting the envelope protein (E) of pan-SARS-CoV, matrix protein (MP) of influenza A virus and non-structural (NS) region of influenza B virus. This quadruplex assay exhibited a sensitivity comparable to its singleplex counterparts and a slightly higher to that of the Centers for Disease Control and Prevention-recommended SARS-CoV-2 and influenza A/B assays. The novel assay showed no false-positive amplifications with other common respiratory viruses, and its 95 % limits of detection for pan-SARS-CoV and influenza A/B virus was 4.26-4.52 copies/reaction. Moreover, the assay was reproducible with less than 1 % coefficient of variation and adaptable testing different clinical and environmental samples. Our ultrafast quadruplex RT-qPCR assay can serve as an attractive tool for effective differentiation of influenza A/B virus and SARS-CoV-2, but more importantly prognose the reemergence/emergence of SARS and novel coronaviruses or influenza viruses from animal spillover.
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Virus de la Influenza A , Virus de la Influenza B , Gripe Humana , Sensibilidad y Especificidad , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza A/clasificación , Virus de la Influenza B/genética , Virus de la Influenza B/aislamiento & purificación , Gripe Humana/virología , Gripe Humana/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex/métodos , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , COVID-19/diagnóstico , COVID-19/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/aislamiento & purificación , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
Bacterial infections in wounds continue to be a major challenge in clinical settings worldwide and represent a significant threat to human health. This work proposes novel expandable and versatile methods for solidifying sodium alginate (SA) with metal ions (such as Fe3+, Co2+, Ni2+, Cu2+, and Zn2+) to create Metal-Alginate (M-Alg) hydrogel with adjustable morphology, composition, and microstructure. It conforms to the wound site, protects against second infection, reduces inflammation, and promotes the healing of infected wounds. Among these hydrogels, Cu-Alginate (Cu-Alg) shows excellent sterilization effect and good efficacy against both gram-positive and gram-negative bacteria, including multidrug-resistant (MDR) strains such as Methicillin-resistant Staphylococcus aureus (MRSA) and Carbapenem-resistant Klebsiella pneumoniae (CRKP) due to its dual antibacterial mechanisms: contact-killing and reactive oxygen species (ROS) burst. Importantly, it exhibits low cytotoxicity and biodegradability. This simple and cost-effective gel-based system has the potential to introduce an innovative approach to the management of wound infection and offers promising new perspectives for the advancement of wound care practice.
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Alginatos , Antibacterianos , Hidrogeles , Staphylococcus aureus Resistente a Meticilina , Infección de Heridas , Alginatos/química , Hidrogeles/química , Hidrogeles/farmacología , Infección de Heridas/tratamiento farmacológico , Infección de Heridas/microbiología , Animales , Antibacterianos/química , Antibacterianos/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones , Klebsiella pneumoniae/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Metales/química , Humanos , Piel/efectos de los fármacos , Piel/microbiología , Especies Reactivas de Oxígeno/metabolismo , Iones/químicaRESUMEN
Euglena gracilis, a model organism of the eukaryotic supergroup Discoba harbouring also clinically important parasitic species, possesses diverse metabolic strategies and an atypical electron transport chain. While structures of the electron transport chain complexes and supercomplexes of most other eukaryotic clades have been reported, no similar structure is currently available for Discoba, limiting the understandings of its core metabolism and leaving a gap in the evolutionary tree of eukaryotic bioenergetics. Here, we report high-resolution cryo-EM structures of Euglena's respirasome I + III2 + IV and supercomplex III2 + IV2. A previously unreported fatty acid synthesis domain locates on the tip of complex I's peripheral arm, providing a clear picture of its atypical subunit composition identified previously. Individual complexes are re-arranged in the respirasome to adapt to the non-uniform membrane curvature of the discoidal cristae. Furthermore, Euglena's conformationally rigid complex I is deactivated by restricting ubiquinone's access to its substrate tunnel. Our findings provide structural insights for therapeutic developments against euglenozoan parasite infections.
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Euglena , Membranas Mitocondriales , Transporte de Electrón , Membranas Mitocondriales/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Metabolismo EnergéticoRESUMEN
The design and fabrication of novel carbon hosts with high conductivity, accelerated electrochemical catalytic activities, and superior physical/chemical confinement on sulfur and its reaction intermediates polysulfides are essential for the construction of high-performance C/S cathodes for lithium-sulfur batteries (LSBs). In this work, a novel biofermentation coupled gel composite assembly technology is developed to prepare cross-linked carbon composite hosts consisting of conductive Rhizopus hyphae carbon fiber (RHCF) skeleton and lamellar sodium alginate carbon (SAC) uniformly implanted with polarized nanoparticles (V2O3, Ag, Co, etc.) with diameters of several nanometers. Impressively, the RHCF/SAC/V2O3 composites exhibit enhanced physical/chemical adsorption of polysulfides due to the synergistic effect between hierarchical pore structures, heteroatoms (N, P) doping, and polar V2O3 generation. Additionally, the catalytic conversion kinetics of cathodes are effectively improved by regulating the 3D carbon structure and optimizing the V2O3 catalyst. Consequently, the LSBs assembled with RHCF/SAC/V2O3-S cathode show exceptional cycle stability (capacity retention rate of 94.0% after 200 cycles at 0.1 C) and excellent rate performance (specific capacity of 578 mA h g-1 at 5 C). This work opens a new door for the fabrication of hyphae carbon composites via fermentation for electrochemical energy storage.
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The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for rapid diagnostic assays to prompt intensified virological monitoring both in human and wild animal populations. To date, there are no clinical validated assays for pan-SARS-coronavirus (pan-SARS-CoV) detection. Here, we suggest an innovative primer design strategy for the diagnosis of pan-SARS-CoVs targeting the envelope (E) gene using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Furthermore, we developed a new primer-probe set targeting human ß2-microglobulin (B2M) as an RNA-based internal control for process efficacy. The universal RT-qPCR assay demonstrated no false-positive amplifications with other human coronaviruses or 20 common respiratory viruses, and its limit of detection (LOD) was 159.16 copies/ml at 95% detection probability. In clinical validation, the assay delivered 100% sensitive results in the detection of SARS-CoV-2-positive oropharyngeal samples (n = 120), including three variants of concern (Wuhan, Delta, and Omicron). Taken together, this universal RT-qPCR assay provides a highly sensitive, robust, and rapid detection of SARS-CoV-1, SARS-CoV-2, and animal-derived SARS-related CoVs.
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Iron is essential for the survival and reproduction of Klebsiella pneumoniae. Although K. pneumoniae employs multiple types of siderophores to scavenge iron during infections, the majority of host iron is retained within erythrocytes and carried by hemoglobin that is inaccessible to siderophores. HmuRSTUV is a bacterial hemin/hemoprotein uptake system. However, the genetic background and function of HmuRSTUV in K. pneumoniae remain unknown. We collected 2,242 K. pneumoniae genomes, of which 2,218 (98.9%) had complete hmuRSTUV loci. Based on the 2,218 complete hmuRSTUV sequences, we established a novel typing scheme of K. pneumoniae named hmST, and 446 nonrepetitive hmSTs were identified. In hypervirulent lineages, hmST was diversely distributed and hmST1 mainly existed in ST23 strains. In contrast, hmST was less diversely distributed among multidrug-resistant strains. hmST demonstrated greater genetic diversity in hypervirulent lineages and community-acquired and bloodstream-sourced strains. In vitro and in vivo experiments revealed that an intact hmuRSTUV was essential for hemin uptake, playing an important role in bloodstream infections. This study established a novel typing scheme of hmST based on hmuRSTUV providing new insights into identifying and monitoring the emergence of novel virulence evolution in K. pneumoniae. IMPORTANCE Siderophore is a group of low molecular weight compounds with high affinity for ferric iron, which could facilitate bacterial iron consumption. Similarly, hemin/heme scavenged by the hemin uptake system HmuRSTUV usually act as another critical iron source for K. pneumoniae. This study proved that Hmu system significantly promoted the growth of K. pneumoniae in the presence of hemin and played an important role in bloodstream infections. A novel typing scheme named hmST was established, and the genetic diversity of hmuRSTUV loci was analyzed based on a large number of genomes. This study provides new insights into identifying and monitoring the emergence of novel virulence evolution in K. pneumoniae.
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BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) infection causes an emerging hemorrhagic fever in East Asia with a high mortality rate. Thrombocytopenia is a consistent feature of SFTS illness, but the mechanism remains elusive. OBJECTIVES: We aimed to better understand the role of platelets in the pathophysiology of SFTSV infection, including the development of thrombocytopenia. METHODS: Using platelets from healthy volunteers and patients with SFTS, we evaluated the functional changes in platelets against SFTSV infection. We investigated the direct effect of glycoprotein VI on platelet-SFTSV interaction by quantitative real-time PCR, molecular docking, surface plasmon resonance spectrometry, flow cytometry, western blot, and platelet functional studies in vitro. Interactions of SFTSV and platelet-SFTSV complexes with macrophages were also determined by scanning electron microscope, quantitative real-time PCR, and flow cytometry. RESULTS: This study is the first to demonstrate that platelets are capable of harboring and producing SFTSV particles. Structural and functional studies found that SFTSVs bind platelet glycoprotein VI to potentiate platelet activation, including platelet aggregation, adenosine triphosphate release, spreading, clot retraction, coagulation, phosphatidylserine exposure, thrombus formation, and adherence. In vitro mechanistic studies highlighted that the interaction of platelets with human THP-1 cells promoted SFTSV clearance and suppressed cytokine production in macrophages. However, unwanted SFTSV replication in macrophages reciprocally aggravated SFTSV persistence in the circulation, which may contribute to thrombocytopenia and other complications during SFTSV infection. CONCLUSION: These findings together highlighted the pathophysiological role of platelets in initial intrinsic defense against SFTSV infections, as well as intertwined processes with host immunity, which can also lead to thrombocytopenia and poor prognosis.
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Infecciones por Bunyaviridae , Síndrome de Trombocitopenia Febril Grave , Trombocitopenia , Humanos , Plaquetas , Síndrome de Trombocitopenia Febril Grave/complicaciones , Infecciones por Bunyaviridae/complicaciones , Simulación del Acoplamiento Molecular , Trombocitopenia/complicaciones , Activación PlaquetariaRESUMEN
Increasing human salmonellosis caused by Salmonella enterica serovar Kentucky and London has raised serious concerns. To better understand possible health risks, insights were provided into specific genetic traits and antimicrobial resistance of 88 representative isolates from human and food sources in Zhejiang Province, China, during 2016-2021. Phylogenomic analysis revealed consistent clustering of isolates into the respective serovar or sequence types, and identified plausible interhost transmission via distinct routes. Each serovar exhibited remarkable diversity in host range and disease-causing potential by cgMLST analyses, and approximately half (48.6%, 17/35) of the food isolates were phylogenetically indistinguishable to those of clinical isolates in the same region. S. London and S. Kentucky harbored serovar-specific virulence genes contributing to their functions in pathogenesis. The overall resistance genotypes correlated with 97.7% sensitivity and 60.2% specificity to the identified phenotypes. Resistance to ciprofloxacin, cefazolin, tetracycline, ampicillin, azithromycin, chloramphenicol, as well as multidrug resistance, was common. High-level dual resistance to ciprofloxacin and cephalosporins in S. Kentucky ST198 isolates highlights evolving threats of antibiotic resistance. These findings underscored the necessity for the development of effective strategies to mitigate the risk of food contamination by Salmonella host-restricted serovars.
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Cefiderocol is a cephalosporin antibiotic presenting expanded antimicrobial activity. CirA is a gateway for cefiderocol to enter bacterial cells. We found that cirA1 and cirA198 were primary alleles in Klebsiella pneumoniae. CirA1 exhibited higher iron-transporting ability than CirA198 in iron-limited conditions. The cefiderocol minimum inhibitory concentration (MIC) increased from 0.5 mg/L to 2 mg/L when cirA1 was mutated to cirA198. Consistently, the MIC showed a 4-fold decrease when cirA198 was mutated to cirA1. Therefore, CirA1 has higher capacity to transport siderophores, contributing to increased cefiderocol susceptibility.
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Klebsiella pneumoniae , Sideróforos , Antibacterianos/farmacología , Cefalosporinas/farmacología , Farmacorresistencia Bacteriana Múltiple , Hierro , Klebsiella pneumoniae/genética , Pruebas de Sensibilidad Microbiana , Sideróforos/farmacología , CefiderocolRESUMEN
[This corrects the article DOI: 10.3389/fphar.2019.00982.].
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Cefiderocol is a novel siderophore cephalosporin exhibiting potent antimicrobial activities. Although cefiderocol has not been approved in China, resistance is emerging. A multicenter study was performed to evaluate the cefiderocol resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP) strains from bloodstream infections in patients with hematologic malignancies in China. Clinical data analysis and whole-genome sequencing were conducted for collected cefiderocol-resistant CRKP strains. CRISPR-Cas9 system was employed to construct site-specific mutagenesis for gene cirA. Plasmid curing and cloning were performed to assess the effect of ß-lactamases on cefiderocol resistance. Total 86 CRKP strains were collected. The MICs of cefiderocol ranged from 0.06 to >256 mg/L. Among four cefiderocol-nonsusceptible strains (4/86, 4.7%), two cefiderocol-resistant strains AR8538 (MIC = 32 mg/L) and AR8416 (MIC > 256 mg/L) were isolated from two patients with acute lymphocytic leukemia (frequency of resistance, 2/86, 2.3%). Metallo- and serine-ß-lactamase inhibitors addition would decrease the MIC of cefiderocol from 32 to 1 mg/L in AR8538, which harbors blaSHV-12, blaDHA-1, and two copies of blaNDM-1 in different plasmids. Avibactam did not impact cefiderocol susceptibility of AR8416, which produces NDM-5. However, we found a deficient CirA in AR8416. Using the same K serotype strain D3, we proved CirA deficiency or carrying NDM individually reduced cefiderocol susceptibility, but their simultaneously existence rendered a high-level cefiderocol resistance. In summary, the resistance of CRKP against cefiderocol is mediated by multiple factors, including the deficiency of CirA, metallo- or serine-ß-lactamases, while a high-level cefiderocol resistance could be rendered by the combined effect of NDM expression and CirA deficiency. IMPORTANCE Cefiderocol-resistant CRKP strains are emerging in bloodstream infections in Chinese patients with hematologic malignancies, although cefiderocol has not been approved for clinical use in China. Our study proved that the resistance of CRKP against cefiderocol is mediated by multiple factors, including the deficiency of CirA, metallo- or serine-ß-lactamases, while a high-level cefiderocol resistance could be rendered by the combined effect of NDM expression and CirA deficiency. As NDM production is one of the most critical mechanisms resulting in carbapenem resistance, it would pose great challenges on the clinical efficacy of cefiderocol in future.
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Neoplasias Hematológicas , Infecciones por Klebsiella , Sepsis , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carbapenémicos , Cefalosporinas/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Neoplasias Hematológicas/complicaciones , Humanos , Klebsiella pneumoniae/genética , Pruebas de Sensibilidad Microbiana , Serina/farmacología , beta-Lactamasas/genética , CefiderocolRESUMEN
The purpose of this study was to assess the risk of aflatoxins due to multiple food consumption among the Zhejiang population. Ultra-high-performance liquid chromatography coupled with tandem mass spectrometry method was used to determine aflatoxins in 792 samples. Aflatoxins were detected in 27.1% of the samples at levels between 0.07 and 262.63 µg kg-1, and aflatoxins B1 was the most frequently detected among different types of samples. 0.8% of peanut oil, 3.39% of nut products as well as 1.1% of condiments contaminated with aflatoxins B1 exceeded China national tolerance limits. Peanut oil had the highest incidence of aflatoxin, with a range from 0.17 to 22.50 µg kg-1. Using bags conferred limited advantages in reducing aflatoxin contents. Moreover, peanut and rice were the main contributors to dietary exposure to aflatoxins among Zhejiang residents. Finally, the margin of exposure values obtained by rice consumption were far from the safe margin of 10,000, indicating a potential risk to public health. The results pointed out the need for further prioritization of aflatoxins B1 risk-management actions in Zhejiang.
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Aflatoxinas , Oryza , Aflatoxina B1/análisis , Arachis/química , China , Cromatografía Líquida de Alta Presión , Contaminación de Alimentos/análisis , Aceite de Cacahuete/análisisRESUMEN
Biotechnology can bring new breakthroughs on design and fabrication of energy materials and devices. In this work, a novel and facile biological self-assembly technology to fabricate multifunctional Rhizopus hyphae carbon fiber (RHCF) and its derivatives on a large scale for electrochemical energy storage is proposed. Crosslinked hollow carbon fibers are successfully prepared by conversion of Rhizopus hyphae, and macroscopic production of centimeter-level carbon balls consisting of hollow RHCFs is further realized. Moreover, the self-assembled RHCF balls show strong adsorption characteristics on metal ions and can be converted into a series of derivatives such as RHCF/metal oxides. Notably, the designed RHCF derivatives are demonstrated with powerful multifunctionability as cathode, anode, and separator for lithium-sulfur batteries (LSBs). The RHCF can act as the host material to combine with metal oxide (CoO) and S, Li metal, and a polypropylene (PP) separator to form a new RHCF/CoO-S cathode, an RHCF/Li anode, and an RHCF/PP separator, respectively. Consequently, the optimized LSB full cell presents excellent cycling performance and superior high-rate capacity (881.3 mA h g-1 at 1 C). This work provides a new method for large-scale preparation of hollow carbon fibers and derivatives for advanced energy storage and conversion.
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OBJECTIVE: Secondary infection, especially bloodstream infection, is an important cause of death in critically ill patients with COVID-19. We aimed to describe secondary bloodstream infection (SBI) in critically ill adults with COVID-19 in the intensive care unit (ICU) and to explore risk factors related to SBI. METHODS: We reviewed all SBI cases among critically ill patients with COVID-19 from 12 February 2020 to 24 March 2020 in the COVID-19 ICU of Jingmen First People's Hospital. We compared risk factors associated with bloodstream infection in this study. All SBIs were confirmed by blood culture. RESULTS: We identified five cases of SBI among the 32 patients: three with Enterococcus faecium, one mixed septicemia (E. faecium and Candida albicans), and one C. parapsilosis. There were no significant differences between the SBI group and non-SBI group. Significant risk factors for SBI were extracorporeal membrane oxygenation, central venous catheter, indwelling urethral catheter, and nasogastric tube. CONCLUSIONS: Our findings confirmed that the incidence of secondary infection, particularly SBI, and mortality are high among critically ill patients with COVID-19. We showed that long-term hospitalization and invasive procedures such as tracheotomy, central venous catheter, indwelling urethral catheter, and nasogastric tube are risk factors for SBI and other complications.
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COVID-19 , Coinfección , Sepsis , Adulto , Enfermedad Crítica , Humanos , Unidades de Cuidados Intensivos , SARS-CoV-2RESUMEN
(1) Background: COVID-19 has been global epidemic. This work aims to extract 3D infection from COVID-19 CT images; (2) Methods: Firstly, COVID-19 CT images are processed with lung region extraction and data enhancement. In this strategy, gradient changes of voxels in different directions respond to geometric characteristics. Due to the complexity of tubular tissues in lung region, they are clustered to the lung parenchyma center based on their filtered possibility. Thus, infection is improved after data enhancement. Then, deep weighted UNet is established to refining 3D infection texture, and weighted loss function is introduced. It changes cost calculation of different samples, causing target samples to dominate convergence direction. Finally, the trained network effectively extracts 3D infection from CT images by adjusting driving strategy of different samples. (3) Results: Using Accuracy, Precision, Recall and Coincidence rate, 20 subjects from a private dataset and eight subjects from Kaggle Competition COVID-19 CT dataset tested this method in hold-out validation framework. This work achieved good performance both in the private dataset (99.94-00.02%, 60.42-11.25%, 70.79-09.35% and 63.15-08.35%) and public dataset (99.73-00.12%, 77.02-06.06%, 41.23-08.61% and 52.50-08.18%). We also applied some extra indicators to test data augmentation and different models. The statistical tests have verified the significant difference of different models. (4) Conclusions: This study provides a COVID-19 infection segmentation technology, which provides an important prerequisite for the quantitative analysis of COVID-19 CT images.