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Genome sequencing is a powerful tool for identifying SARS-CoV-2 variant lineages; however, there can be limitations due to sequence dropout when used to identify specific key mutations. Recently, ThermoFisher Scientific has developed genotyping assays to help bridge the gap between testing capacity and sequencing capability to generate real-time genotyping results based on specific variants. Over a 6-week period during the months of April and May 2021, we set out to assess the ThermoFisher TaqMan mutation panel genotyping assay, initially for three mutations of concern and then for an additional two mutations of concern, against SARS-CoV-2-positive clinical samples and the corresponding COVID-19 Genomics UK Consortium (COG-UK) sequencing data. We demonstrate that genotyping is a powerful in-depth technique for identifying specific mutations, is an excellent complement to genome sequencing, and has real clinical health value potential, allowing laboratories to report and take action on variants of concern much more quickly.
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COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Prueba de COVID-19 , Humanos , Mutación , SARS-CoV-2/genéticaRESUMEN
A SARS-CoV-2 variant B1.1.7 containing mutation Δ69/70 has spread rapidly in the United Kingdom and shows an identifiable profile in ThermoFisher TaqPath RT-qPCR, S gene target failure (SGTF). We analyzed recent test data for trends and significance. Linked cycle threshold (Ct) values for respiratory samples showed that a low Ct for ORF1ab and N were clearly associated with SGTF. Significantly more SGTF samples had higher inferred viral loads between 1×107 and 1×108. Our conclusion is that patients whose samples exhibit the SGTF profile are more likely to have high viral loads, which may explain higher infectivity and rapidity of spread.
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COVID-19/virología , Reacción en Cadena de la Polimerasa/métodos , SARS-CoV-2/fisiología , Carga Viral , COVID-19/epidemiología , Humanos , Modelos Lineales , Reacción en Cadena de la Polimerasa/normas , SARS-CoV-2/clasificación , SARS-CoV-2/genética , Polimerasa TaqRESUMEN
In mice, the IgG subclass induced after Ag encounter can reflect the nature of the Ag. Th2 Ags such as alum-precipitated proteins and helminths induce IgG1, whereas Th1 Ags, such as Salmonella Typhimurium, predominantly induce IgG2a. The contribution of different IgG isotypes to protection against bacteria such as S. Typhimurium is unclear, although as IgG2a is induced by natural infection, it is assumed this isotype is important. Previously, we have shown that purified S. Typhimurium porins including outer membrane protein OmpD, which induce both IgG1 and IgG2a in mice, provide protection to S. Typhimurium infection via Ab. In this study we report the unexpected finding that mice lacking IgG1, but not IgG2a, are substantially less protected after porin immunization than wild-type controls. IgG1-deficient mice produce more porin-specific IgG2a, resulting in total IgG levels that are similar to wild-type mice. The decreased protection in IgG1-deficient mice correlates with less efficient bacterial opsonization and uptake by macrophages, and this reflects the low binding of outer membrane protein OmpD-specific IgG2a to the bacterial surface. Thus, the Th2-associated isotype IgG1 can play a role in protection against Th1-associated organisms such as S. Typhimurium. Therefore, individual IgG subclasses to a single Ag can provide different levels of protection and the IgG isotype induced may need to be a consideration when designing vaccines and immunization strategies.
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Anticuerpos Antibacterianos/inmunología , Inmunoglobulina G/inmunología , Porinas/inmunología , Vacunas contra la Salmonella/inmunología , Salmonella typhimurium/inmunología , Animales , Reacciones Antígeno-Anticuerpo , Adhesión Bacteriana/inmunología , Proteínas Bacterianas/inmunología , Línea Celular , Femenino , Deficiencia de IgG/inmunología , Inmunización , Cambio de Clase de Inmunoglobulina , Isotipos de Inmunoglobulinas/inmunología , Masculino , Ratones Endogámicos C57BL , Fagocitosis/inmunología , Salmonelosis Animal/prevención & controlRESUMEN
INTRODUCTION: Infection causes a vast burden of disease, with significant mortality, morbidity and costs to health-care systems. However, identifying the pathogen causative infection can be challenging, resulting in high use of broad-spectrum antibiotics, much of which may be inappropriate. Novel metagenomic methods have potential to rapidly identify pathogens, however their clinical utility for many infections is currently unclear. Outcome from infection is also impacted by the effectiveness of immune responses, which can be impaired by age, co-morbidity and the infection itself. The aims of this study are twofold: To compare diversity of organisms identified and time-to-result using metagenomic methods versus traditional culture -based techniques, to explore the potential clinical role of metagenomic approaches to pathogen identification in a range of infections.To characterise the ex vivo function of immune cells from patients with acute infection, exploring host and pathogen-specific factors which may affect immune function and overall outcomes. METHODS: This is a prospective observational study of patients with acute infection. Patients with symptoms suggestive of an acute infection will be recruited, and blood and bodily fluid relevant to the site of infection collected (for example, sputum and naso-oropharyngeal swabs for respiratory tract infections, or urine for a suspected urinary tract infection). Metagenomic analysis of samples will be compared to traditional microbiology, alongside the antimicrobials received. Blood and respiratory samples such as bronchoalveolar lavage will be used to isolate immune cells and interrogate immune cell function. Where possible, similar samples will be collected from matched participants without a suspected infection to determine the impact of infection on both microbiome and immune cell function.
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Antibacterianos , Infecciones del Sistema Respiratorio , Humanos , Antibacterianos/uso terapéutico , Lavado Broncoalveolar , Proyectos de Investigación , Infecciones del Sistema Respiratorio/diagnóstico , Metagenómica , Estudios Observacionales como AsuntoRESUMEN
The risk to human health from mosquito-borne viruses such as dengue, chikungunya and yellow fever is increasing due to increased human expansion, deforestation and climate change. To anticipate and predict the spread and transmission of mosquito-borne viruses, a better understanding of the transmission cycle in mosquito populations is needed. We present a pathogen-agnostic combined sequencing protocol for identifying vectors, viral pathogens and their hosts or reservoirs using portable Oxford Nanopore sequencing. Using mosquitoes collected in São Paulo, Brazil, we extracted RNA for virus identification and DNA for blood meal and mosquito identification. Mosquitoes and blood meals were identified by comparing cytochrome c oxidase I (COI) sequences against a curated Barcode of Life Data System (BOLD). Viruses were identified using the SMART-9N protocol, which allows amplified DNA to be prepared with native barcoding for nanopore sequencing. Kraken 2 was employed to detect viral pathogens and Minimap2 and BOLD identified the contents of the blood meal. Due to the high similarity of some species, mosquito identification was conducted using blast after generation of consensus COI sequences using RACON polishing. This protocol can simultaneously uncover viral diversity, mosquito species and mosquito feeding habits. It also has the potential to increase understanding of mosquito genetic diversity and transmission dynamics of zoonotic mosquito-borne viruses.
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Arbovirus , Culicidae , Secuenciación de Nanoporos , Animales , Humanos , Culicidae/genética , Arbovirus/genética , Mosquitos Vectores , Brasil , ADNRESUMEN
Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicemia. The ability of this organism to survive inside phagocytic cells is poorly understood but thought to be an important step for the establishment of disease in the host. Here, we demonstrate that GBS shows prolonged survival within J774 macrophages and that the capacity to survive is not significantly changed across a diverse range of strains representing different serotypes, multilocus sequence types (MLST), and sites of clinical isolation. Using staining for the lysosome-associated membrane protein (LAMP) and by pharmacological inhibition of phagosome acidification, we demonstrate that streptococci reside in a phagosome and that acidification of the phagosome is required for GBS to survive intracellularly. Moreover, we show that the GBS two-component system CovS/CovR, which is the major acid response regulator in this organism, is required for survival inside the phagosome.
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Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Macrófagos/microbiología , Proteínas Represoras/metabolismo , Streptococcus agalactiae/genética , Streptococcus agalactiae/fisiología , Animales , Cápsulas Bacterianas , Proteínas Bacterianas/genética , Línea Celular , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Histidina Quinasa , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/fisiología , Ratones , Perforina/genética , Perforina/metabolismo , Fagosomas , Pigmentos Biológicos , Especies Reactivas de Oxígeno , Proteínas Represoras/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Long-term severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in immunodeficient patients are an important source of variation for the virus but are understudied. Many case studies have been published which describe one or a small number of long-term infected individuals but no study has combined these sequences into a cohesive dataset. This work aims to rectify this and study the genomics of this patient group through a combination of literature searches as well as identifying new case series directly from the COVID-19 Genomics UK (COG-UK) dataset. The spike gene receptor-binding domain and N-terminal domain (NTD) were identified as mutation hotspots. Numerous mutations associated with variants of concern were observed to emerge recurrently. Additionally a mutation in the envelope gene, T30I was determined to be the second most frequent recurrently occurring mutation arising in persistent infections. A high proportion of recurrent mutations in immunodeficient individuals are associated with ACE2 affinity, immune escape, or viral packaging optimisation. There is an apparent selective pressure for mutations that aid cell-cell transmission within the host or persistence which are often different from mutations that aid inter-host transmission, although the fact that multiple recurrent de novo mutations are considered defining for variants of concern strongly indicates that this potential source of novel variants should not be discounted.
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BACKGROUND: Multidrug-resistant Acinetobacter baumannii commonly causes hospital outbreaks. However, within an outbreak, it can be difficult to identify the routes of cross-infection rapidly and accurately enough to inform infection control. Here, we describe a protracted hospital outbreak of multidrug-resistant A. baumannii, in which whole-genome sequencing (WGS) was used to obtain a high-resolution view of the relationships between isolates. METHODS: To delineate and investigate the outbreak, we attempted to genome-sequence 114 isolates that had been assigned to the A. baumannii complex by the Vitek2 system and obtained informative draft genome sequences from 102 of them. Genomes were mapped against an outbreak reference sequence to identify single nucleotide variants (SNVs). RESULTS: We found that the pulsotype 27 outbreak strain was distinct from all other genome-sequenced strains. Seventy-four isolates from 49 patients could be assigned to the pulsotype 27 outbreak on the basis of genomic similarity, while WGS allowed 18 isolates to be ruled out of the outbreak. Among the pulsotype 27 outbreak isolates, we identified 31 SNVs and seven major genotypic clusters. In two patients, we documented within-host diversity, including mixtures of unrelated strains and within-strain clouds of SNV diversity. By combining WGS and epidemiological data, we reconstructed potential transmission events that linked all but 10 of the patients and confirmed links between clinical and environmental isolates. Identification of a contaminated bed and a burns theatre as sources of transmission led to enhanced environmental decontamination procedures. CONCLUSIONS: WGS is now poised to make an impact on hospital infection prevention and control, delivering cost-effective identification of routes of infection within a clinically relevant timeframe and allowing infection control teams to track, and even prevent, the spread of drug-resistant hospital pathogens.
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OBJECTIVES: Pseudomonas aeruginosa is a common nosocomial pathogen responsible for significant morbidity and mortality internationally. Patients may become colonised or infected with P. aeruginosa after exposure to contaminated sources within the hospital environment. The aim of this study was to determine whether whole-genome sequencing (WGS) can be used to determine the source in a cohort of burns patients at high risk of P. aeruginosa acquisition. STUDY DESIGN: An observational prospective cohort study. SETTING: Burns care ward and critical care ward in the UK. PARTICIPANTS: Patients with >7% total burns by surface area were recruited into the study. METHODS: All patients were screened for P. aeruginosa on admission and samples taken from their immediate environment, including water. Screening patients who subsequently developed a positive P. aeruginosa microbiology result were subject to enhanced environmental surveillance. All isolates of P. aeruginosa were genome sequenced. Sequence analysis looked at similarity and relatedness between isolates. RESULTS: WGS for 141 P. aeruginosa isolates were obtained from patients, hospital water and the ward environment. Phylogenetic analysis revealed eight distinct clades, with a single clade representing the majority of environmental isolates in the burns unit. Isolates from three patients had identical genotypes compared with water isolates from the same room. There was clear clustering of water isolates by room and outlet, allowing the source of acquisitions to be unambiguously identified. Whole-genome shotgun sequencing of biofilm DNA extracted from a thermostatic mixer valve revealed this was the source of a P. aeruginosa subpopulation previously detected in water. In the remaining two cases there was no clear link to the hospital environment. CONCLUSIONS: This study reveals that WGS can be used for source tracking of P. aeruginosa in a hospital setting, and that acquisitions can be traced to a specific source within a hospital ward.