Next generation sequencing (NGS) is increasingly being used in clinical microbiology. Like every new technology adopted in microbiology, the integration of NGS into clinical and routine workflows ...must be carefully managed.
To review the practical aspects of implementing bacterial whole genome sequencing (WGS) in routine diagnostic laboratories.
Review of the literature and expert opinion.
In this review, we discuss when and how to integrate whole genome sequencing (WGS) in the routine workflow of the clinical laboratory. In addition, as the microbiology laboratories have to adhere to various national and international regulations and criteria for their accreditation, we deliberate on quality control issues for using WGS in microbiology, including the importance of proficiency testing. Furthermore, the current and future place of this technology in the diagnostic hierarchy of microbiology is described as well as the necessity of maintaining backwards compatibility with already established methods. Finally, we speculate on the question of whether WGS can entirely replace routine microbiology in the future and the tension between the fact that most sequencers are designed to process multiple samples in parallel whereas for optimal diagnosis a one-by-one processing of the samples is preferred. Special reference is made to the cost and turnaround time of WGS in diagnostic laboratories.
Further development is required to improve the workflow for WGS, in particular to shorten the turnaround time, reduce costs, and streamline downstream data analyses. Only when these processes reach maturity will reliance on WGS for routine patient management and infection control management become feasible, enabling the transformation of clinical microbiology into a genome-based and personalized diagnostic field.
Next-generation sequencing (NGS) is transforming microbiology. With the increased accessibility and decrease in the costs of sequencing and the optimisation of the 'wet laboratory' components of NGS ...i.e. the quality and throughput of DNA extraction, library preparation and sequencing reactions, whole genome sequencing (WGS) of bacterial isolates is rapidly revolutionising clinical and public health microbiology. WGS is a 'disruptive technology' that has the potential to become a one-stop-shop for routine bacterial analysis.
Microbial whole genome sequencing (WGS) has many advantages over standard microbiological methods. However, it is not yet widely implemented in routine hospital diagnostics due to notable challenges.
...The aim was to extract managerial, financial and clinical criteria supporting the decision to implement WGS in routine diagnostic microbiology, across different operational models of implementation in the hospital setting.
This was a systematic review of literature identified through PubMed and Web of Science. English literature studies discussing the applications of microbial WGS without limitation on publication date were eligible. A narrative approach for categorization and synthesis of the sources identified was adopted.
A total of 98 sources were included. Four main alternative operational models for incorporating WGS in clinical microbiology laboratories were identified: full in-house sequencing and analysis, full outsourcing of sequencing and analysis and two hybrid models combining in-house/outsourcing of the sequencing and analysis components. Six main criteria (and multiple related sub-criteria) for WGS implementation emerged from our review and included cost (e.g. the availability of resources for capital and operational investment); manpower (e.g. the ability to provide training programmes or recruit trained personnel), laboratory infrastructure (e.g. the availability of supplies and consumables or sequencing platforms), bioinformatics requirements (e.g. the availability of valid analysis tools); computational infrastructure (e.g. the availability of storage space or data safety arrangements); and quality control (e.g. the existence of standardized procedures).
The decision to incorporate WGS in routine diagnostics involves multiple, sometimes competing, criteria and sub-criteria. Mapping these criteria systematically is an essential stage in developing policies for adoption of this technology, e.g. using a multicriteria decision tool. Future research that will prioritize criteria and sub-criteria that were identified in our review in the context of operational models will inform decision-making at clinical and managerial levels with respect to effective implementation of WGS for routine use. Beyond WGS, similar decision-making challenges are expected with respect to future integration of clinical metagenomics.
Shotgun sequencing is increasingly applied in clinical microbiology for unbiased culture-independent diagnosis. While software solutions for metagenomics proliferate, integration of metagenomics in ...clinical care requires method standardization and validation. Virtual metagenomics samples could underpin validation by substituting real samples and thus we sought to develop a novel solution for simulation of metagenomics samples based on user-defined clinical scenarios.
We designed the Microbial Metagenomics Mock Scenario-based Sample Simulation (M3S3) workflow, which allows users to generate virtual samples from raw reads or assemblies. The M3S3 output is a mock sample in FASTQ or FASTA format. M3S3 was tested by generating virtual samples for 10 challenging infectious disease scenarios, involving a background matrix ‘spiked’ in silico with pathogens including mixtures. Replicate samples (seven per scenario) were used to represent different compositional ratios. Virtual samples were analysed using Taxonomer and Kraken db.
The 10 challenge scenarios were successfully applied, generating 80 samples. For all tested scenarios, the virtual samples showed sequence compositions as predicted from the user input. Spiked pathogen sequences were identified with the majority of the replicates and most exhibited acceptable abundance (deviation between expected and observed abundance of spiked pathogens), with slight differences observed between software tools.
Despite demonstrated proof-of-concept, integration of clinical metagenomics in routine microbiology remains a substantial challenge. M3S3 is capable of producing virtual samples on-demand, simulating a spectrum of clinical diagnostic scenarios of varying complexity. The M3S3 tool can therefore support the development and validation of standardized metagenomics applications.
Presently, the bottleneck in the deployment of high-throughput sequencing technology is the ability to analyse the increasing amount of data produced in a fit-for-purpose manner. The field of ...microbial bioinformatics is thriving and quickly adapting to technological changes, which creates difficulties for nonbioinformaticians in following the complexity and increasingly obscure jargon of this field.
This review is directed towards nonbioinformaticians who wish to gain understanding of the overall microbial bioinformatic processes, from raw data obtained from sequencers to final outputs.
The software and analytical strategies reviewed are based on the personal experience of the authors.
The bioinformatic processes of transforming raw reads to actionable information in a clinical and epidemiologic context is explained. We review the advantages and limitations of two major strategies currently applied: read mapping, which is the comparison with a predefined reference genome, and de novo assembly, which is the unguided assembly of the raw data. Finally, we discuss the main analytical methodologies and the most frequently used freely available software and its application in the context of bacterial infectious disease management.
High-throughput sequencing technologies are overhauling outbreak investigation and epidemiologic surveillance while creating new challenges due to the amount and complexity of data generated. The continuously evolving field of microbial bioinformatics is required for stakeholders to fully harness the power of these new technologies.
The term 'Diagnostic stewardship' is increasingly being used in the literature. This term appears to have emerged in order to address two related and topical aspects of the role of the microbiology ...laboratory in clinical practice today: i) using microbiology diagnostic tests to improve the use of antibiotics, and ii) promoting the appropriate use of microbiology diagnostic methods. While 'Diagnostic stewardship' is catchy, we worry that it may ultimately be a misleading term with the potential to impede communication with non-infection specialists. We present here the views of two ESCMID study group executive committees (ESGMD and ESGAP) with the hope of stimulating further discussion.
Crohn's disease (CD) is a chronic inflammatory bowel disease associated with psychological distress and intestinal microbial changes. Here, we examined whether a 3-month period of Cognitive ...Behavioral and Mindfulness with Daily Exercise (COBMINDEX) intervention, which improves the wellbeing and inflammatory state of CD patients, may also affect their gut microbiome. Gut microbiota, circulating inflammatory markers and hormones were analyzed in 24 CD patients before (T1) and after 3 months of COBMINDEX (T2), and in 25 age- and sex-matched wait-list control patients at the corresponding time-points. Microbiota analysis examined relative taxonomical abundance, alpha and beta diversity, and microbiome correlations with inflammatory and psychological parameters. At T1, CD patients exhibited a characteristic microbial profile mainly constituted of Proteobacteria (17.71%), Firmicutes (65.56%), Actinobacteria (8.46%) and Bacteroidetes (6.24%). Baseline bacterial abundances showed significant correlations with psychological markers of distress and with IFN
. Following COBMINDEX, no significant changes in alpha and beta diversity were observed between both study groups, though a trend change in beta diversity was noted. Significant changes occurred in the abundance of phyla, families and genera only among the COBMINDEX group. Furthermore, abundance of phyla, families and genera that were altered following COBMNIDEX, significantly correlated with levels of cytokines and psychological parameters. Our results demonstrated that a short-term intervention of COBMINDEX was associated with changes in microbial indices, some of which are linked to psychological manifestations and systemic inflammation in CD patients. Psychological interventions to reduce chronic stress, such as COBMINDEX, appear to be beneficial in mitigating the pathobiology of CD patients, and may thus provide a useful adjunct to pharmacological therapy.
During the winter period 2010/11 27 epidemiologically unlinked, confirmed cases of oseltamivir-resistant influenza A(H1N1)2009 virus infection have been detected in multiple, geographically dispersed ...settings. Three of these cases were in community settings, with no known exposure to oseltamivir. This suggests possible onward transmission of resistant strains and could be an indication of a possibility of changing epidemiology of oseltamivir-resistant influenza A(H1N1)2009 virus.
Israel was certified as polio-free country in June 2002, along with the rest of the World Health Organization European Region. Some 11 years later, wild-type polio virus ₁ (WPV₁) was isolated ...initially from routine sewage samples collected between 7 and 13 April 2013 in two cities in the Southern district. WPV₁-specific analysis of samples indicated WPV₁ introduction into that area in early February 2013. National supplementary immunisation with oral polio vaccine has been ongoing since August 2013.
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