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The COVID-19 pandemic led to a large global effort to sequence SARS-CoV-2 genomes from patient samples to track viral evolution and inform public health response. Millions of SARS-CoV-2 genome sequences have been deposited in global public repositories. The Canadian COVID-19 Genomics Network (CanCOGeN - VirusSeq), a consortium tasked with coordinating expanded sequencing of SARS-CoV-2 genomes across Canada early in the pandemic, created the Canadian VirusSeq Data Portal, with associated data pipelines and procedures, to support these efforts. The goal of VirusSeq was to allow open access to Canadian SARS-CoV-2 genomic sequences and enhanced, standardized contextual data that were unavailable in other repositories and that meet FAIR standards (Findable, Accessible, Interoperable and Reusable). In addition, the Portal data submission pipeline contains data quality checking procedures and appropriate acknowledgement of data generators that encourages collaboration. From inception to execution, the portal was developed with a conscientious focus on strong data governance principles and practices. Extensive efforts ensured a commitment to Canadian privacy laws, data security standards, and organizational processes. This Portal has been coupled with other resources like Viral AI and was further leveraged by the Coronavirus Variants Rapid Response Network (CoVaRR-Net) to produce a suite of continually updated analytical tools and notebooks. Here we highlight this Portal, including its contextual data not available elsewhere, and the 'Duotang', a web platform that presents key genomic epidemiology and modeling analyses on circulating and emerging SARS-CoV-2 variants in Canada. Duotang presents dynamic changes in variant composition of SARS-CoV-2 in Canada and by province, estimates variant growth, and displays complementary interactive visualizations, with a text overview of the current situation. The VirusSeq Data Portal and Duotang resources, alongside additional analyses and resources computed from the Portal (COVID-MVP, CoVizu), are all open-source and freely available. Together, they provide an updated picture of SARS-CoV-2 evolution to spur scientific discussions, inform public discourse, and support communication with and within public health authorities. They also serve as a framework for other jurisdictions interested in open, collaborative sequence data sharing and analyses.
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SETTING: Early in the COVID-19 pandemic, the Public Health Agency of Canada (PHAC) and provincial/territorial (P/T) public health identified the need for a coordinated response to complex multijurisdictional COVID-19 outbreaks. The first large multijurisdictional industrial worksite COVID-19 outbreak highlighted the risk of transmission within these congregate work settings, the risk of transmission to the broader community(ies), and the need to develop setting-specific outbreak response frameworks. INTERVENTION: PHAC assembled a team to provide national outbreak support for multijurisdictional COVID-19 outbreaks in May 2020. The COVID-19 Outbreak Response Unit (ORU) worked with P/T partners to develop guiding principles for outbreak response and outbreak investigation processes, guidance documents, and investigation tools (e.g., minimum data elements and questionnaires). OUTCOMES: The ORU, P/T partners, and onsite industrial worksite health and safety staff leveraged outbreak investigation guidelines, industrial worksite outbreak process documents (including minimum data elements), and enhanced case questionnaires to respond to multiple COVID-19 outbreak investigations in industrial worksites. Clear roles/responsibilities and processes, along with standardized data, allowed for more efficient outbreak investigations and earlier implementation of mitigation measures. IMPLICATIONS: Multijurisdictional COVID-19 outbreaks highlighted the importance of public health collaboration with industry partners onsite. The assembly of a national outbreak response team was important to facilitate information sharing and provide technical support. Lessons learned and recommendations on outbreak preparation, detection, management, and communication are included to enhance a response framework applicable to future emerging or re-emerging pathogens with epidemic and/or pandemic potential.
RéSUMé: CONTEXTE: Au début de la pandémie de COVID-19, l'Agence de la santé publique du Canada (ASPC) et les autorités provinciales/territoriales de santé publique ont reconnu la nécessité d'une réponse coordonnée en cas d'éclosions complexes multi-juridictionnelles de COVID-19. La première grande éclosion multi-juridictionnelle de COVID-19 dans un chantier industriel a mis en évidence le risque de transmission dans ces milieux de travail collectifs, le risque de transmission à l'ensemble de la (des) communauté(s) et la nécessité d'élaborer des cadres d'intervention en cas d'éclosion spécifiques aux types de milieux. INTERVENTION: L'ASPC a formé une équipe chargée de soutenir la réponse nationale contre les éclosions multi-juridictionnelles de COVID-19 en mai 2020. L'Unité d'intervention en cas d'éclosion (UIE) de COVID-19 a collaboré avec des partenaires provinciaux et territoriaux pour élaborer des principes de référence pour la lutte contre les éclosions de COVID-19 et des processus d'enquête sur les éclosions, des documents d'orientation et des outils d'enquête (p.ex. des éléments de données minimales et des questionnaires). RéSULTATS: L'UIE, les provinces et territoires et le personnel chargé de la santé et sécurité du travail sur le site se sont appuyés sur des principes de référence aux enquêtes sur les éclosions, les documents de processus d'enquête sur les éclosions dans les sites industriels, y compris les éléments de données minimales et le questionnaire détaillé sur les cas, pour répondre à multiples enquêtes d'éclosions de COVID-19 dans les sites industriels. Des rôles/responsabilités et des processus clairs, ainsi que des données standardisées, ont permis de mener des enquêtes plus efficaces sur les éclosions et de mettre en Åuvre plus rapidement des mesures d'atténuation. IMPLICATIONS: Les éclosions multi-juridictionnelles de COVID-19 ont mis en évidence l'importance de la collaboration entre les autorités de santé publique et les partenaires industriels sur site. La constitution d'une équipe nationale d'intervention en cas d'éclosion a été importante pour faciliter le partage des informations et fournir un soutien technique. Les connaissances acquises et les recommandations sur la préparation, la détection, la gestion et la communication des éclosions sont incluses afin d'améliorer le cadre de réponse aux futurs agents pathogènes émergents ou ré-émergents ayant un potentiel épidémique et/ou pandémique.
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COVID-19 , Surtos de Doenças , Local de Trabalho , Humanos , COVID-19/epidemiologia , COVID-19/prevenção & controle , Canadá/epidemiologia , Surtos de Doenças/prevenção & controle , Acampamento , Indústrias , Saúde OcupacionalRESUMO
Fast, efficient public health actions require well-organized and coordinated systems that can supply timely and accurate knowledge. Public databases of pathogen genomic data, such as the International Nucleotide Sequence Database Collaboration (INSDC), have become essential tools for efficient public health decisions. However, these international resources began primarily for academic purposes, rather than for surveillance or interventions. Now, queries need to access not only the whole genomes of multiple pathogens but also make connections using robust contextual metadata to identify issues of public health relevance. Databases that over time developed a patchwork of submission formats and requirements need to be consistently organized and coordinated internationally to allow effective searches.To help resolve these issues, we propose a common pathogen data structure called the Pathogen Data Object Model (DOM) that will formalize the minimum pieces of sequence data and contextual data necessary for general public health uses, while recognizing that submitters will likely withhold a wide range of non-public contextual data. Further, we propose contributors use the Pathogen DOM for all pathogen submissions (bacterial, viral, fungal, and parasites), which will simplify data submissions and provide a consistent and transparent data structure for downstream data analyses. We also highlight how improved submission tools can support the Pathogen DOM, offering users additional easy-to-use methods to ensure this structure is followed.
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Nucleotídeos , Saúde Pública , Sequência de Bases , Genômica/métodos , Bases de Dados de Ácidos NucleicosRESUMO
From pathogen detection to genome or plasmid closure, the utility of the Oxford Nanopore Technologies (ONT) MinION for microbiological analysis has been well documented. The MinION's small footprint, portability, and real-time analytic capability situates it well to address challenges in the field of unbiased pathogen detection, as a component of a security investigation. To this end, a multicenter evaluation of the effect of alternative analytical approaches on the outcome of MinION-based sequencing, using a set of well-characterized samples, was explored in a field-based scenario. Three expert scientific response groups evaluated known bacterial DNA extracts as part of an international first responder (Chemical, Biological, Radiological) training exercise. Samples were prepared independently for analysis using the Rapid and/or Rapid PCR sequencing kits as per the best practices of each of the participating groups. Analyses of sequence data were in turn conducted using varied approaches including ONTs What's in my pot (WIMP) architecture and in-house computational pipelines. Microbial community composition and the ability of each approach to detect pathogens was compared. Each group demonstrated the ability to detect all species present in samples, although several organisms were detected at levels much lower than expected with some organisms even falling below 1% abundance. Several 'contaminant' near neighbor species were also detected, at low abundance. Regardless of the sequencing approach chosen, the observed composition of the bacterial communities diverged from the input composition in each of the analyses, although sequencing conducted using the rapid kit produced the least distortion when compared to PCR-based library preparation methods. One of the participating groups generated drastically lower sequencing output than the other groups, likely attributed to the limited computer hard drive capacity, and occasional disruption of the internet connection. These results provide further consideration for conducting unbiased pathogen identification within a field setting using MinION sequencing. However, the benefits of this approach in providing rapid results and unbiased detection must be considered along with the complexity of sample preparation and data analytics, when compared to more traditional methods. When utilized by trained scientific experts, with appropriate computational resources, the MinION sequencing device is a useful tool for field-based pathogen detection in mixed samples.
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Sequenciamento por Nanoporos , Nanoporos , Análise de Sequência de DNA/métodos , Bactérias/genética , Genoma , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
BACKGROUND: The reliability of culture-independent pathogen detection in foods using metagenomics is contingent on the quality and composition of the reference database. The inclusion of microbial sequences from a diverse representation of taxonomies in universal reference databases is recommended to maximize classification precision for pathogen detection. However, these sizable databases have high memory requirements that may be out of reach for some users. In this study, we aimed to assess the performance of a foodborne pathogen (FBP)-specific reference database (taxon-specific) relative to a universal reference database (taxon-agnostic). We tested our FBP-specific reference database's performance for detecting Listeria monocytogenes in two complex food matrices-ready-to-eat (RTE) turkey deli meat and prepackaged spinach-using three popular read-based DNA-to-DNA metagenomic classifiers: Centrifuge, Kraken 2 and KrakenUniq. RESULTS: In silico host sequence removal led to substantially fewer false positive (FP) classifications and higher classification precision in RTE turkey deli meat datasets using the FBP-specific reference database. No considerable improvement in classification precision was observed following host filtering for prepackaged spinach datasets and was likely a consequence of a higher microbe-to-host sequence ratio. All datasets classified with Centrifuge using the FBP-specific reference database had the lowest classification precision compared to Kraken 2 or KrakenUniq. When a confidence-scoring threshold was applied, a nearly equivalent precision to the universal reference database was achieved for Kraken 2 and KrakenUniq. Recall was high for both reference databases across all datasets and classifiers. Substantially fewer computational resources were required for metagenomics-based detection of L. monocytogenes using the FBP-specific reference database, especially when combined with Kraken 2. CONCLUSIONS: A universal (taxon-agnostic) reference database is not essential for accurate and reliable metagenomics-based pathogen detection of L. monocytogenes in complex food matrices. Equivalent classification performance can be achieved using a taxon-specific reference database when the appropriate quality control measures, classification software, and analysis parameters are applied. This approach is less computationally demanding and more attainable for the broader scientific and food safety communities.
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Listeria monocytogenes , Listeria monocytogenes/genética , Spinacia oleracea , Microbiologia de Alimentos , Metagenômica , Reprodutibilidade dos Testes , CarneRESUMO
Pathogen genomics is a critical tool for public health surveillance, infection control, outbreak investigations as well as research. In order to make use of pathogen genomics data, they must be interpreted using contextual data (metadata). Contextual data include sample metadata, laboratory methods, patient demographics, clinical outcomes and epidemiological information. However, the variability in how contextual information is captured by different authorities and how it is encoded in different databases poses challenges for data interpretation, integration and their use/re-use. The DataHarmonizer is a template-driven spreadsheet application for harmonizing, validating and transforming genomics contextual data into submission-ready formats for public or private repositories. The tool's web browser-based JavaScript environment enables validation and its offline functionality and local installation increases data security. The DataHarmonizer was developed to address the data sharing needs that arose during the COVID-19 pandemic, and was used by members of the Canadian COVID Genomics Network (CanCOGeN) to harmonize SARS-CoV-2 contextual data for national surveillance and for public repository submission. In order to support coordination of international surveillance efforts, we have partnered with the Public Health Alliance for Genomic Epidemiology to also provide a template conforming to its SARS-CoV-2 contextual data specification for use worldwide. Templates are also being developed for One Health and foodborne pathogens. Overall, the DataHarmonizer tool improves the effectiveness and fidelity of contextual data capture as well as its subsequent usability. Harmonization of contextual information across authorities, platforms and systems globally improves interoperability and reusability of data for concerted public health and research initiatives to fight the current pandemic and future public health emergencies. While initially developed for the COVID-19 pandemic, its expansion to other data management applications and pathogens is already underway.
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COVID-19 , Humanos , COVID-19/epidemiologia , Pandemias , SARS-CoV-2/genética , Canadá , Genômica/métodosRESUMO
BACKGROUND: The advent of metagenomic sequencing provides microbial abundance patterns that can be leveraged for sample origin prediction. Supervised machine learning classification approaches have been reported to predict sample origin accurately when the origin has been previously sampled. Using metagenomic datasets provided by the 2019 CAMDA challenge, we evaluated the influence of variable technical, analytical and machine learning approaches for result interpretation and novel source prediction. RESULTS: Comparison between 16S rRNA amplicon and shotgun sequencing approaches as well as metagenomic analytical tools showed differences in normalized microbial abundance, especially for organisms present at low abundance. Shotgun sequence data analyzed using Kraken2 and Bracken, for taxonomic annotation, had higher detection sensitivity. As classification models are limited to labeling pre-trained origins, we took an alternative approach using Lasso-regularized multivariate regression to predict geographic coordinates for comparison. In both models, the prediction errors were much higher in Leave-1-city-out than in 10-fold cross validation, of which the former realistically forecasted the increased difficulty in accurately predicting samples from new origins. This challenge was further confirmed when applying the model to a set of samples obtained from new origins. Overall, the prediction performance of the regression and classification models, as measured by mean squared error, were comparable on mystery samples. Due to higher prediction error rates for samples from new origins, we provided an additional strategy based on prediction ambiguity to infer whether a sample is from a new origin. Lastly, we report increased prediction error when data from different sequencing protocols were included as training data. CONCLUSIONS: Herein, we highlight the capacity of predicting sample origin accurately with pre-trained origins and the challenge of predicting new origins through both regression and classification models. Overall, this work provides a summary of the impact of sequencing technique, protocol, taxonomic analytical approaches, and machine learning approaches on the use of metagenomics for prediction of sample origin.
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Metagenoma , Metagenômica/métodos , Microbiota/genética , RNA Ribossômico 16S/análise , Aprendizado de Máquina SupervisionadoRESUMO
INTRODUCTION: Pouchitis is a common complication after ileal pouch-anal anastomosis (IPAA). However, there is a poor correlation between symptoms and endoscopic appearance of the pouch, and many patients can have debilitating symptoms in the absence of overt inflammation. It is unknown whether these clinical symptoms are independently associated with the microbiota. The objective of this work was to examine whether the individual clinical components of the pouch activity scoring systems are associated with specific microbiota. METHODS: Pouch biopsies from 233 patients (50% male, 100% IPAA/ulcerative colitis) post-IPAA were included. Clinical phenotyping was performed, and patients were classified using both clinical and endoscopic components of the Pouch Activity Scale. Scoring for symptoms examined 24-hour stool frequency, urgency, incontinence, and rectal bleeding as described by the Pouchitis Disease Activity Index Score. RESULTS: In the absence of inflammation, an increase in stool frequency reported over 24 hours was associated with a decrease in Bacteroidetes relative abundance, and this was the strongest association found. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis in inflamed groups showed that an increase in 24-hour stool frequency was associated with an increase in biofilm formation. DISCUSSION: These findings indicate that in patients with IPAA, the composition of mucosa-associated microbiota of the pouch may contribute to clinical symptoms, particularly stool frequency, independent of endoscopic disease activity.
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Bolsas Cólicas/microbiologia , Microbioma Gastrointestinal/imunologia , Íleo/microbiologia , Mucosa Intestinal/microbiologia , Pouchite/diagnóstico , Proctocolectomia Restauradora/efeitos adversos , Adulto , Idoso , Biópsia , Colite Ulcerativa/microbiologia , Colite Ulcerativa/cirurgia , Bolsas Cólicas/patologia , Colonoscopia , Feminino , Seguimentos , Humanos , Íleo/diagnóstico por imagem , Íleo/patologia , Íleo/cirurgia , Mucosa Intestinal/diagnóstico por imagem , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Pouchite/imunologia , Pouchite/microbiologia , Índice de Gravidade de DoençaRESUMO
The MinION sequencer (Oxford Nanopore Technologies) is a paradigm shifting device allowing rapid, real time long read sequencing of nucleic acids. Yet external benchmarking of this technologies' capabilities has not been extensively reported, nor has thorough evaluation of its utility for field-based analysis with sub-optimal sample types been described. The aim of this study was to evaluate the capability of the MinION sequencer for bacterial genomic and metagenomic applications, with specific emphasis placed on the quality, yield, and accuracy of generated sequence data. Two independent laboratories at the National Microbiology Laboratory (Public Health Agency of Canada), sequenced a set of microbes in replicate, using the currently available flowcells, sequencing chemistries, and software available at the time of the experiment. Overall sequencing yield and quality improved through the course of this set of experiments. Sequencing alignment accuracy was high reaching 97% for all 2D experiments, though was slightly lower for 1D sequencing (94%). 1D sequencing provided much longer sequences than 2D. Both sequencing chemistries performed equally well in constructing genomic assemblies. There was evidence of barcode cross-over using both the native and PCR barcoding methods. Despite the sub-optimal nature of samples sequenced in the field, sequences attributable to B. anthracis the target organism used in this scenario, could none-the-less be detected. Together, this report showcases the rapid advancement in this technology and its utility in the context of genomic sequencing of microbial isolates of importance to public health.
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Bacillus anthracis/genética , Sequenciamento Completo do Genoma/instrumentação , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Metagenômica , NanoporosRESUMO
Canada has one of the lowest rates of tuberculosis (TB) in the world, however, among certain sub-populations, disease incidence rates approach those observed in sub-Saharan Africa, and other high incidence regions. In this study, we applied mycobacterial interspersed repetitive unit (MIRU) variable number of tandem repeat (VNTR) and whole genome sequencing (WGS) to the analysis of Mycobacterium tuberculosis isolates obtained from Northern communities in the territory of Nunavut. WGS was carried out using the Illumina MiSeq, with identified variants used to infer phylogenetic relationships and annotated to infer functional implications. Additionally, the sequencing data from these isolates were augmented with publically available WGS to evaluate data from the Nunavut outbreak in the broader Canadian context. In this study, isolates could be classified into four major clusters by MIRU-VNTR analysis. These could be further resolved into sub-clusters using WGS. No evidence for antimicrobial resistance, either genetic or phenotypic, was observed in this cohort. Among most subjects with multiple samples, reactivation/incomplete treatment likely contributed to recurrence. However, isolates from two subjects appeared more likely to have occurred via reinfection, based on the large number of genomic single nucleotide variants detected. Finally, although quite distinct from previously reported Canadian MTB strains, isolates obtained from Nunavut clustered most closely with a cohort of samples originating in the Nunavik region of Northern Quebec. This study demonstrates the benefit of using WGS for discriminatory analysis of MTB in Canada, especially in high incidence regions. It further emphasizes the importance of focusing epidemiological intervention efforts on interrupting transmission chains of endemic TB throughout Northern communities, rather than relying on strategies applied in regions where the majority of TB cases result from importation of foreign strains.
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Genoma Bacteriano , Mycobacterium tuberculosis/genética , Tuberculose/epidemiologia , Estudos de Coortes , Surtos de Doenças , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Nunavut/epidemiologia , Tuberculose/microbiologiaRESUMO
A high-quality custom database of MALDI-TOF mass spectral profiles was developed with the goal of improving clinical diagnostic identification of high-consequence bacterial pathogens. A biomarker discovery method is presented for identifying and evaluating MALDI-TOF MS spectra to potentially differentiate biothreat bacteria from less-pathogenic near-neighbour species.
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Bactérias/isolamento & purificação , Bactérias/patogenicidade , Técnicas de Tipagem Bacteriana , Biomarcadores/análise , Bases de Dados de Ácidos Nucleicos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Bacillus/genética , Bacillus/isolamento & purificação , Bactérias/genética , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , Proteínas de Bactérias/isolamento & purificação , Armas Biológicas , Brucella/genética , Brucella/isolamento & purificação , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normas , Yersinia/genética , Yersinia/isolamento & purificaçãoRESUMO
Mutations in the erm(41) gene of M.abscessus group organisms are associated with differences in inducible macrolide resistance, with current recommendations being to hold rapidly growing isolates for up to 14 days in order to ensure that resistance which develops more slowly can be detected. This study aimed to determine the ideal incubation time for accurate identification of inducible macrolide resistance as well as to determine if there was an association between the time taken to detect inducible resistance in M.abscessus group organisms and their erm(41) sequevar. We amplified and sequenced the erm(41) genes of a total of 104 M.abscessus group isolates and determined their sequevars. The isolates were tested for phenotypic clarithromycin resistance at days 7, 10, 14 and 21, using Trek Diagnostics Sensititre RAPMYCO microbroth dilution plates. Associations between erm(41) gene sequevars and time to detection of resistance were evaluated using Fisher's exact test in R. The samples included in this study fell into 14 sequevars, with the majority of samples falling into Sequevar02 (16), Sequevar06 (15), Sequevar08 (7) and Sequvar 15 (31), and several isolates that were in small clusters, or unique. The majority (82.7%) of samples exhibiting inducible macrolide resistance were interpreted as resistant by day 7. Two isolates in Sequevar02, which has a T28C mutation that is associated with sensitivity, showed intermediate resistance at day 14, though the majority (13) were sensitive at day 14. The majority of isolates with inducible macrolide resistance fell into Sequevars 06,08 and 15, none of which contain the T28C mutation. These sequevars were analyzed to determine if there was any correlation between sequevar and time to detection of resistance. None was found. Based on these findings, we recommend the addition of a day 7 read to the CLSI guidelines to improve turn-around-times for these isolates. It is also recommended that erm(41) gene sequencing be added to routine phenotypic testing for the resolution of cases with difficult-to-interpret phenotypic results.
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Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Macrolídeos/farmacologia , Mycobacterium/genética , Claritromicina/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Metiltransferases/genética , Testes de Sensibilidade Microbiana , Mycobacterium/efeitos dos fármacos , Mycobacterium/isolamento & purificação , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Análise de Sequência de DNA , Fatores de TempoRESUMO
Following restorative proctocolectomy with an ileal pouch-anal anastomosis, the small bowel mucosa undergoes several specific histologic adaptions, which may be unrelated to the underlying disease or symptoms of pouchitis. An increase in intraepithelial lymphocytes (IELs) has not been described as part of this spectrum. Mucosal biopsies of the ileal pouch and afferent limb of 230 patients (mean age: 45.7y [18.3-74.7], gender [female/male]: 117/113) with a functioning ileal pouch-anal anastomosis (mean time since ileostomy closure: 10.8months) and associated clinically annotated outcome data were assessed for IELs/100 enterocytes. Forty-two patients (18.3%) showed an increase in IELs (≥20 IELs/100 enterocytes [range 20-39]), in pouch and/or afferent limb biopsies. Intraepithelial lymphocytosis was more commonly observed in afferent limb compared to pouch biopsies (18.8% vs 8.3%; P = .42) and in familial adenomatous polyposis compared to ulcerative colitis patients (16% vs 8%; P = 0.36), but neither difference reached statistical significance. No cases with increased IELs displayed severe villous blunting. Increased IELs were not significantly associated with age, sex, ethnicity, smoking history, time since ileostomy, use of antibiotics, biologic agents, anti-diarrheal agents or probiotics, C-reactive protein levels or differential white cell count. None of the 42 patients with increased IELs had positive celiac serology (anti-human tissue transglutaminase IgA [ELISA] with corresponding total serum IgA). Intraepithelial lymphocytosis in pouch biopsies may represent a subclinical response to an altered bacterial microenvironment. Pathologists should be aware that intraepithelial lymphocytosis is part of the spectrum of changes in pouch biopsies, and only rarely is due to celiac disease.
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Polipose Adenomatosa do Colo/cirurgia , Colite Ulcerativa/cirurgia , Bolsas Cólicas/efeitos adversos , Enterócitos/patologia , Íleo/patologia , Linfocitose/patologia , Pouchite/patologia , Polipose Adenomatosa do Colo/diagnóstico , Adolescente , Adulto , Idoso , Biópsia , Doença Celíaca/patologia , Colite Ulcerativa/diagnóstico , Diagnóstico Diferencial , Feminino , Humanos , Linfocitose/etiologia , Masculino , Pessoa de Meia-Idade , Pouchite/etiologia , Valor Preditivo dos Testes , Testes Sorológicos , Resultado do Tratamento , Adulto JovemRESUMO
Inflammatory bowel disease has been associated with differential abundance of numerous organisms when compared to healthy controls (HCs); however, few studies have investigated variability in the microbiome across intestinal locations and how this variability might be related to disease location and phenotype. In this study, we have analyzed the microbiome of a large cohort of individuals recruited at Mount Sinai Hospital in Toronto, Canada. Biopsies were taken from subjects with Crohn's disease, ulcerative colitis, and HC, and also individuals having undergone ileal pouch-anal anastomosis for treatment of ulcerative colitis or familial adenomatous polyposis. Microbial 16S rRNA was sequenced using the Illumina MiSeq platform. We observed a great deal of variability in the microbiome characterizing different sampling locations. Samples from pouch and afferent limb were comparable in microbial composition. When comparing sigmoid and terminal ileum samples, more differences were observed. The greatest number of differentially abundant microbes was observed when comparing either pouch or afferent limb samples to sigmoid or terminal ileum. Despite these differences, we were able to observe modest microbial variability between inflammatory bowel disease phenotypes and HCs, even when controlling for sampling location and additional experimental factors. Most detected associations were observed between HCs and Crohn's disease, with decreases in specific genera in the families Ruminococcaceae and Lachnospiraceae characterizing tissue samples from individuals with Crohn's disease. This study highlights important considerations when analyzing the composition of the microbiome and also provides useful insight into differences in the microbiome characterizing these seemingly related phenotypes.
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Colite Ulcerativa/microbiologia , Bolsas Cólicas/microbiologia , Doença de Crohn/microbiologia , Íleo/microbiologia , Intestinos/microbiologia , Microbiota , Adulto , Canadá , Estudos de Casos e Controles , Estudos de Coortes , Colite Ulcerativa/patologia , Colite Ulcerativa/cirurgia , Bolsas Cólicas/patologia , Doença de Crohn/patologia , Doença de Crohn/cirurgia , Feminino , Seguimentos , Humanos , Íleo/patologia , Intestinos/patologia , Masculino , Pessoa de Meia-Idade , Fenótipo , PrognósticoRESUMO
Whereas the infant gut microbiome is the subject of intense study, relatively little is known regarding the nares microbiome in newborns and during early life. This study aimed to survey the typical composition and diversity of human anterior nare microflora for developing infants over time, and to explore how these correlate to their primary caregivers. Single nare swabs were collected at five time points over a one-year period for each subject from infant-caregiver pairs. Our study comprised of 50 infants (recruited at 2 weeks, post delivery) and their 50 primary caregivers. Applying the chaperonin-60 (cpn60) universal target (UT) amplicon as our molecular barcoding marker to census survey the microbial communities, we longitudinally surveyed infant nares microbiota at 5 time points over the course of the first year of life. The inter- and intra-subject diversity was catalogued and compared, both longitudinally and relative to their adult primary caregivers. Although within-subject variability over time and inter-subject variability were both observed, the assessment detected only one or two predominant genera for individual infant samples, belonging mainly to phyla Actinobacteria, Firmicutes, and Proteobacteria. Consistent with previously observed microbial population dynamics in other body sites, the diversity of nares microflora increased over the first year of life and infants showed differential operational taxonomic units (OTUs) relative to their matched primary caregiver. The collected evidence also support that both temporal and seasonal changes occur with respect to carriage of potentially pathogenic bacteria (PPBs), which may influence host predisposition to infection. This pilot study surveying paired infant/caregiver nare microbiomes provides novel longitudinal diversity information that is pertinent to better understanding nare microbiome development in infants.
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Cuidadores , Chaperonina 60/genética , Microbiota/genética , Nariz/microbiologia , Filogenia , Adulto , Biodiversidade , Marcadores Genéticos , Humanos , Lactente , Recém-Nascido , Análise de Sequência de DNARESUMO
The advent and widespread application of next-generation sequencing (NGS) technologies to the study of microbial genomes has led to a substantial increase in the number of studies in which whole genome sequencing (WGS) is applied to the analysis of microbial genomic epidemiology. However, microorganisms such as Mycobacterium tuberculosis (MTB) present unique problems for sequencing and downstream analysis based on their unique physiology and the composition of their genomes. In this study, we compare the quality of sequence data generated using the Nextera and TruSeq isolate preparation kits for library construction prior to Illumina sequencing-by-synthesis. Our results confirm that MTB NGS data quality is highly dependent on the purity of the DNA sample submitted for sequencing and its guanine-cytosine content (or GC-content). Our data additionally demonstrate that the choice of library preparation method plays an important role in mitigating downstream sequencing quality issues. Importantly for MTB, the Illumina TruSeq library preparation kit produces more uniform data quality than the Nextera XT method, regardless of the quality of the input DNA. Furthermore, specific genomic sequence motifs are commonly missed by the Nextera XT method, as are regions of especially high GC-content relative to the rest of the MTB genome. As coverage bias is highly undesirable, this study illustrates the importance of appropriate protocol selection when performing NGS studies in order to ensure that sound inferences can be made regarding mycobacterial genomes.
Assuntos
Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mycobacterium tuberculosis , Manejo de Espécimes/métodos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificaçãoRESUMO
BACKGROUND: Inflammatory pouch complications refractory to first-line therapies remain problematic following ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC). We evaluated infliximab efficacy and associations with therapeutic response. METHODS: Data from individuals who underwent colectomy and IPAA for UC (2000-2014) were reviewed. Patients with chronic refractory pouchitis (CP) and Crohn's disease (CD)-like outcomes treated with infliximab were included. Pre-treatment parameters and response at median 8 (initial) and 48 weeks (sustained) were measured. Complete response was defined as symptomatic and endoscopic resolution with modified Pouchitis Disease Activity Index (mPDAI) <5. Partial response included mPDAI improvement >2. Serum was analysed for Anti-Saccharomyces cerevisiae antibodies (ASCA), anti-OmpC, anti-CBir1 and perinuclear Anti-Neutrophil Cytoplasmic Antibodies (pANCA). RESULTS: One hundred and fifty-two patients with CP or a CD-like phenotype were identified. Forty-two were treated with infliximab (33% male; age 32.6±2.6 years, 28.5% CD-like). Post-induction response was achieved in 74% (48% complete) and sustained response in 62.6% (29.6% complete). Mean mPDAI and C-reactive protein declined from 8.5±0.3 to 2±3.4 (p < 0.002) and from 29.48±6.2 to 5.76±1.6mg/L (p < 0.001), respectively. Female gender, smoking and presence of anti-CBir1 were associated with infliximab use (p < 0.01) but not response. Pre-treatment mPDAI <10 (p < 0.01), resolution of rectal bleeding (p < 0.001 ) and week 8 endoscopic activity were associated with sustained response (p = 0.04; odds ratio [OR] 2.2; 95% confidence interval [CI] 1.1-16.5]). More than 2 positive antimicrobial antibody titres were associated with non-response (p < 0.05), but did not retain significance in multivariate analysis (p = 0.197; OR 0.632; 95% CI 0.31-1.2). CONCLUSIONS: Infliximab can effectively treat inflammatory pouch complications. Pre-treatment mPDAI <10 and early endoscopy may identify responders.
Assuntos
Colite Ulcerativa/tratamento farmacológico , Bolsas Cólicas/efeitos adversos , Fármacos Gastrointestinais/uso terapêutico , Infliximab/uso terapêutico , Pouchite/tratamento farmacológico , Adulto , Colite Ulcerativa/cirurgia , Feminino , Humanos , Masculino , Estudos Retrospectivos , Resultado do TratamentoRESUMO
BACKGROUND: Pouchitis is common after ileal pouch-anal anastomosis (IPAA) surgery for ulcerative colitis (UC). Similar to inflammatory bowel disease (IBD), both host genetics and the microbiota are implicated in its pathogenesis. We use the IPAA model of IBD to associate mucosal host gene expression with mucosal microbiomes and clinical outcomes. We analyze host transcriptomic data and 16S rRNA gene sequencing data from paired biopsies from IPAA patients with UC and familial adenomatous polyposis. To achieve power for a genome-wide microbiome-transcriptome association study, we use principal component analysis for transcript and clade reduction, and identify significant co-variation between clades and transcripts. RESULTS: Host transcripts co-vary primarily with biopsy location and inflammation, while microbes co-vary primarily with antibiotic use. Transcript-microbe associations are surprisingly modest, but the most strongly microbially-associated host transcript pattern is enriched for complement cascade genes and for the interleukin-12 pathway. Activation of these host processes is inversely correlated with Sutterella, Akkermansia, Bifidobacteria, and Roseburia abundance, and positively correlated with Escherichia abundance. CONCLUSIONS: This study quantifies the effects of inflammation, antibiotic use, and biopsy location upon the microbiome and host transcriptome during pouchitis. Understanding these effects is essential for basic biological insights as well as for well-designed and adequately-powered studies. Additionally, our study provides a method for profiling host-microbe interactions with appropriate statistical power using high-throughput sequencing, and suggests that cross-sectional changes in gut epithelial transcription are not a major component of the host-microbiome regulatory interface during pouchitis.
Assuntos
Bolsas Cólicas/microbiologia , Microbioma Gastrointestinal , Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Doenças Inflamatórias Intestinais/microbiologia , Mucosa/microbiologia , Adolescente , Adulto , Idoso , Estudos de Coortes , Colite Ulcerativa/genética , Colite Ulcerativa/microbiologia , Bolsas Cólicas/patologia , Feminino , Trato Gastrointestinal/microbiologia , Perfilação da Expressão Gênica , Humanos , Doenças Inflamatórias Intestinais/cirurgia , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Análise Multivariada , Pouchite/genética , Pouchite/microbiologia , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/isolamento & purificação , Análise de Sequência de DNA , Adulto JovemRESUMO
The development of culture-independent techniques and next-generation sequencing has led to a staggering rise in the number of microbiome studies over the last decade. Although it remains important to identify the taxa of microbes present in a variety of environmental samples, including the gut microbiomes of healthy and diseased individuals, the next stage of microbiome research will need to focus on uncovering the role of the microbiome rather than its mere composition. Here, we introduce techniques that go beyond identifying the taxa present within a sample and examine the biological function of the microbiome or the host-microbiome interaction.
RESUMO
Inflammatory bowel disease (IBD) is an abnormal inflammatory response within the gut to a trigger that has yet to be identified. The family history in many patients, especially those with Crohn's disease, suggests a genetic predisposition. It has been hypothesized that the abnormal inflammatory response is due in part to genetic alterations in the normal homeostatic processes that regulate host interactions with the normal gut microbes. Genetic studies have identified increasing numbers of genetic risk factors that involve a diverse series of pathways such as receptors of innate immune response, defects in epithelial barrier function, immune- and cytokine-related genes and genes involved in autophagy. Studies further suggest that abnormal immune responses in IBD patients are directed against the intestinal microbiota, with activation of both innate and adaptive immune responses. Indeed, studies have shown bacterial-derived antigen as drivers of T cell immune responses. More recently, Th17, regulatory T cells and unconventional innate-like T cells have been implicated in the induction and regulation of intestinal inflammation. The seminal discoveries of pathogen recognition receptors including Toll-like receptors and nucleotide-binding oligomerization domain receptors have changed our understanding of how immune cells respond to microbes and the role this may play in IBD pathogenesis. Understanding these mechanism will lead to new strategies in the treatment and prevention of IBD.