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      Set-up and validation of mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) analysis of Mycobacterium tuberculosis using BioNumerics software

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          Abstract

          The objective was to describe and validate a new and alternative software procedure for 24-locus mycobacterial interspersed repetitive unit-variable number-tandem repeat (MIRU-VNTR) typing of Mycobacterium tuberculosis (Mtb) based on the multipurpose BioNumerics software. DNA from randomly selected isolates of Mtb from two European laboratories, including external control samples for MIRU-VNTR typing, were analysed. Samples were genotyped using the commercial 24-locus VNTR typing kit from GenoScreen. The PCR amplified fragments were separated by capillary electrophoresis. For the subsequent analyses, the currently used software GeneMapper was compared with BioNumerics. The endpoint was the level of concordance when comparing genotyping results obtained from BioNumerics with results obtained from GeneMapper and the ECDC proficiency study reference results. Also, the number of necessary manual standard size corrections and allele assignments in the two different software methods were compared. In total, 272 DNA samples, including the ECDC proficiency panel, were analysed. For all samples, there were 100% concordance of results. For a randomly selected set of 96 samples the numbers of manual corrections needed for size standards were 199 with GeneMapper versus zero for BioNumerics. The numbers of manual corrections for allele assignments were 122 with GeneMapper versus 16 with BioNumerics. In conclusion, we have validated the multipurpose software BioNumerics for standard 24-locus MIRU-VNTR typing and the software shows promising benefits in terms of simplification and minimization of hand-on time.

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          Implementation of Whole Genome Sequencing (WGS) for Identification and Characterization of Shiga Toxin-Producing Escherichia coli (STEC) in the United States

          Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen capable of causing severe disease in humans. Rapid and accurate identification and characterization techniques are essential during outbreak investigations. Current methods for characterization of STEC are expensive and time-consuming. With the advent of rapid and cheap whole genome sequencing (WGS) benchtop sequencers, the potential exists to replace traditional workflows with WGS. The aim of this study was to validate tools to do reference identification and characterization from WGS for STEC in a single workflow within an easy to use commercially available software platform. Publically available serotype, virulence, and antimicrobial resistance databases were downloaded from the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org) and integrated into a genotyping plug-in with in silico PCR tools to confirm some of the virulence genes detected from WGS data. Additionally, down sampling experiments on the WGS sequence data were performed to determine a threshold for sequence coverage needed to accurately predict serotype and virulence genes using the established workflow. The serotype database was tested on a total of 228 genomes and correctly predicted from WGS for 96.1% of O serogroups and 96.5% of H serogroups identified by conventional testing techniques. A total of 59 genomes were evaluated to determine the threshold of coverage to detect the different WGS targets, 40 were evaluated for serotype and virulence gene detection and 19 for the stx gene subtypes. For serotype, 95% of the O and 100% of the H serogroups were detected at > 40x and ≥ 30x coverage, respectively. For virulence targets and stx gene subtypes, nearly all genes were detected at > 40x, though some targets were 100% detectable from genomes with coverage ≥20x. The resistance detection tool was 97% concordant with phenotypic testing results. With isolates sequenced to > 40x coverage, the different databases accurately predicted serotype, virulence, and resistance from WGS data, providing a fast and cheaper alternative to conventional typing techniques.
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            First worldwide proficiency study on variable-number tandem-repeat typing of Mycobacterium tuberculosis complex strains.

            Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra- and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter- and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future.
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              Molecular surveillance of multi- and extensively drug-resistant tuberculosis transmission in the European Union from 2003 to 2011.

              The European Centre for Disease Prevention and Control (ECDC) initiated a project on the molecular surveillance of multi- and extensively drug-resistant tuberculosis (MDR-/XDR-TB) transmission in the European Union (EU) in the period from 2009 to 2011. In total, 2,092 variable number of tandem repeat (VNTR) patterns of MDR-/XDR-TB Mycobacterium tuberculosis isolates were collected, originating from 24 different countries in the period 2003 to 2011. Of the collected VNTR patterns, 45% (n=941) could be assigned to one of the 79 European multiple-country molecular fingerprint clusters and 50% of those (n=470) belonged to one extremely large cluster caused by Beijing strains of one genotype. We conclude that international transmission of MDR-/XDR-TB plays an important role in the EU, especially in the eastern part, and is significantly related to the spread of one strain or clone of the Beijing genotype. Implementation of international cluster investigation in EU countries should reveal underlying factors of transmission, and show how TB control can be improved regarding case finding, contact tracing, infection control and treatment in order to prevent further spread of MDR-/XDR-TB in the EU.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                31 October 2018
                2018
                : 13
                : 10
                : e0205336
                Affiliations
                [1 ] International Reference Laboratory of Mycobacteriology, Statens Serum Institut, Copenhagen, Denmark
                [2 ] Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark
                [3 ] Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019—UMR 8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Lille, France
                [4 ] Genoscreen, Campus de l'Institut Pasteur de Lille, Lille, France
                Institut de Pharmacologie et de Biologie Structurale, FRANCE
                Author notes

                Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Philip Supply is a consultant for GenoScreen. The other authors have no competing interests to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

                Author information
                http://orcid.org/0000-0002-7469-5963
                Article
                PONE-D-18-00069
                10.1371/journal.pone.0205336
                6209162
                30379832
                78a66da7-ad93-422f-a4de-e3923a0d93c0
                © 2018 Pedersen et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 21 February 2018
                : 24 September 2018
                Page count
                Figures: 0, Tables: 0, Pages: 5
                Funding
                Philip Supply is a consultant for GenoScreen. The funder provided support in the form of salaries for author PS but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Bacteria
                Actinobacteria
                Mycobacterium Tuberculosis
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Genotyping
                Research and Analysis Methods
                Molecular Biology Techniques
                Genotyping
                Medicine and Health Sciences
                Infectious Diseases
                Bacterial Diseases
                Tuberculosis
                Medicine and Health Sciences
                Tropical Diseases
                Tuberculosis
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                People and Places
                Geographical Locations
                Europe
                Research and Analysis Methods
                Electrophoretic Techniques
                Capillary Electrophoresis
                Medicine and Health Sciences
                Infectious Diseases
                Bacterial Diseases
                Tuberculosis
                Multi-Drug-Resistant Tuberculosis
                Medicine and Health Sciences
                Tropical Diseases
                Tuberculosis
                Multi-Drug-Resistant Tuberculosis
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Genome Sequencing
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Genome Sequencing
                Custom metadata
                Data are available from Mendeley ( http://dx.doi.org/10.17632/z975xz4bk4.1).

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