TY - JOUR T1 - Closing the gap: Oxford Nanopore Technologies R10 sequencing allows comparable results to Illumina sequencing for SNP-based outbreak investigation of bacterial pathogens. JF - J Clin Microbiol Y1 - 2024 A1 - Bogaerts, Bert ED - Van den Bossche, An ED - Verhaegen, Bavo ED - Laurence Delbrassinne ED - Wesley Mattheus ED - Nouws, Stéphanie ED - Godfroid, Maxime ED - Hoffman, Stefan ED - Nancy Roosens ED - De Keersmaecker, Sigrid C J ED - Vanneste, Kevin KW - Illumina KW - nanopore sequencing KW - outbreak KW - public health KW - whole-genome sequencing. AB -

Whole-genome sequencing has become the method of choice for bacterial outbreak investigation, with most clinical and public health laboratories currently routinely using short-read Illumina sequencing. Recently, long-read Oxford Nanopore Technologies (ONT) sequencing has gained prominence and may offer advantages over short-read sequencing, particularly with the recent introduction of the R10 chemistry, which promises much lower error rates than the R9 chemistry. However, limited information is available on its performance for bacterial single-nucleotide polymorphism (SNP)-based outbreak investigation. We present an open-source workflow, Prokaryotic Awesome variant Calling Utility (PACU) (https://github.com/BioinformaticsPlatformWIV-ISP/PACU), for constructing SNP phylogenies using Illumina and/or ONT R9/R10 sequencing data. The workflow was evaluated using outbreak data sets of Shiga toxin-producing and by comparing ONT R9 and R10 with Illumina data. The performance of each sequencing technology was evaluated not only separately but also by integrating samples sequenced by different technologies/chemistries into the same phylogenomic analysis. Additionally, the minimum sequencing time required to obtain accurate phylogenetic results using nanopore sequencing was evaluated. PACU allowed accurate identification of outbreak clusters for both species using all technologies/chemistries, but ONT R9 results deviated slightly more from the Illumina results. ONT R10 results showed trends very similar to Illumina, and we found that integrating data sets sequenced by either Illumina or ONT R10 for different isolates into the same analysis produced stable and highly accurate phylogenomic results. The resulting phylogenies for these two outbreaks stabilized after ~20 hours of sequencing for ONT R9 and ~8 hours for ONT R10. This study provides a proof of concept for using ONT R10, either in isolation or in combination with Illumina, for rapid and accurate bacterial SNP-based outbreak investigation.

M3 - 10.1128/jcm.01576-23 ER - TY - JOUR T1 - Closing the gap: Oxford Nanopore Technologies R10 sequencing allows comparable results to Illumina sequencing for SNP-based outbreak investigation of bacterial pathogens. JF - J Clin Microbiol Y1 - 2024 A1 - Bogaerts, Bert ED - Van den Bossche, An ED - Verhaegen, Bavo ED - Laurence Delbrassinne ED - Wesley Mattheus ED - Nouws, Stéphanie ED - Godfroid, Maxime ED - Hoffman, Stefan ED - Nancy Roosens ED - De Keersmaecker, Sigrid C J ED - Vanneste, Kevin KW - Illumina KW - nanopore sequencing KW - outbreak KW - public health KW - whole-genome sequencing. AB -

Whole-genome sequencing has become the method of choice for bacterial outbreak investigation, with most clinical and public health laboratories currently routinely using short-read Illumina sequencing. Recently, long-read Oxford Nanopore Technologies (ONT) sequencing has gained prominence and may offer advantages over short-read sequencing, particularly with the recent introduction of the R10 chemistry, which promises much lower error rates than the R9 chemistry. However, limited information is available on its performance for bacterial single-nucleotide polymorphism (SNP)-based outbreak investigation. We present an open-source workflow, Prokaryotic Awesome variant Calling Utility (PACU) (https://github.com/BioinformaticsPlatformWIV-ISP/PACU), for constructing SNP phylogenies using Illumina and/or ONT R9/R10 sequencing data. The workflow was evaluated using outbreak data sets of Shiga toxin-producing and by comparing ONT R9 and R10 with Illumina data. The performance of each sequencing technology was evaluated not only separately but also by integrating samples sequenced by different technologies/chemistries into the same phylogenomic analysis. Additionally, the minimum sequencing time required to obtain accurate phylogenetic results using nanopore sequencing was evaluated. PACU allowed accurate identification of outbreak clusters for both species using all technologies/chemistries, but ONT R9 results deviated slightly more from the Illumina results. ONT R10 results showed trends very similar to Illumina, and we found that integrating data sets sequenced by either Illumina or ONT R10 for different isolates into the same analysis produced stable and highly accurate phylogenomic results. The resulting phylogenies for these two outbreaks stabilized after ~20 hours of sequencing for ONT R9 and ~8 hours for ONT R10. This study provides a proof of concept for using ONT R10, either in isolation or in combination with Illumina, for rapid and accurate bacterial SNP-based outbreak investigation.

M3 - 10.1128/jcm.01576-23 ER - TY - JOUR T1 - Closing the gap: Oxford Nanopore Technologies R10 sequencing allows comparable results to Illumina sequencing for SNP-based outbreak investigation of bacterial pathogens. JF - J Clin Microbiol Y1 - 2024 A1 - Bogaerts, Bert ED - Van den Bossche, An ED - Verhaegen, Bavo ED - Laurence Delbrassinne ED - Wesley Mattheus ED - Nouws, Stéphanie ED - Godfroid, Maxime ED - Hoffman, Stefan ED - Nancy Roosens ED - De Keersmaecker, Sigrid C J ED - Vanneste, Kevin KW - Illumina KW - nanopore sequencing KW - outbreak KW - public health KW - whole-genome sequencing. AB -

Whole-genome sequencing has become the method of choice for bacterial outbreak investigation, with most clinical and public health laboratories currently routinely using short-read Illumina sequencing. Recently, long-read Oxford Nanopore Technologies (ONT) sequencing has gained prominence and may offer advantages over short-read sequencing, particularly with the recent introduction of the R10 chemistry, which promises much lower error rates than the R9 chemistry. However, limited information is available on its performance for bacterial single-nucleotide polymorphism (SNP)-based outbreak investigation. We present an open-source workflow, Prokaryotic Awesome variant Calling Utility (PACU) (https://github.com/BioinformaticsPlatformWIV-ISP/PACU), for constructing SNP phylogenies using Illumina and/or ONT R9/R10 sequencing data. The workflow was evaluated using outbreak data sets of Shiga toxin-producing and by comparing ONT R9 and R10 with Illumina data. The performance of each sequencing technology was evaluated not only separately but also by integrating samples sequenced by different technologies/chemistries into the same phylogenomic analysis. Additionally, the minimum sequencing time required to obtain accurate phylogenetic results using nanopore sequencing was evaluated. PACU allowed accurate identification of outbreak clusters for both species using all technologies/chemistries, but ONT R9 results deviated slightly more from the Illumina results. ONT R10 results showed trends very similar to Illumina, and we found that integrating data sets sequenced by either Illumina or ONT R10 for different isolates into the same analysis produced stable and highly accurate phylogenomic results. The resulting phylogenies for these two outbreaks stabilized after ~20 hours of sequencing for ONT R9 and ~8 hours for ONT R10. This study provides a proof of concept for using ONT R10, either in isolation or in combination with Illumina, for rapid and accurate bacterial SNP-based outbreak investigation.

M3 - 10.1128/jcm.01576-23 ER - TY - JOUR T1 - Closing the gap: Oxford Nanopore Technologies R10 sequencing allows comparable results to Illumina sequencing for SNP-based outbreak investigation of bacterial pathogens. JF - J Clin Microbiol Y1 - 2024 A1 - Bert Bogaerts A1 - An Van den Bossche A1 - Bavo Verhaegen A1 - Laurence Delbrassinne A1 - Wesley Mattheus A1 - Stéphanie Nouws A1 - Maxime Godfroid A1 - Stefan Hoffman A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker A1 - Kevin Vanneste AB -

Whole-genome sequencing has become the method of choice for bacterial outbreak investigation, with most clinical and public health laboratories currently routinely using short-read Illumina sequencing. Recently, long-read Oxford Nanopore Technologies (ONT) sequencing has gained prominence and may offer advantages over short-read sequencing, particularly with the recent introduction of the R10 chemistry, which promises much lower error rates than the R9 chemistry. However, limited information is available on its performance for bacterial single-nucleotide polymorphism (SNP)-based outbreak investigation. We present an open-source workflow, Prokaryotic Awesome variant Calling Utility (PACU) (https://github.com/BioinformaticsPlatformWIV-ISP/PACU), for constructing SNP phylogenies using Illumina and/or ONT R9/R10 sequencing data. The workflow was evaluated using outbreak data sets of Shiga toxin-producing and by comparing ONT R9 and R10 with Illumina data. The performance of each sequencing technology was evaluated not only separately but also by integrating samples sequenced by different technologies/chemistries into the same phylogenomic analysis. Additionally, the minimum sequencing time required to obtain accurate phylogenetic results using nanopore sequencing was evaluated. PACU allowed accurate identification of outbreak clusters for both species using all technologies/chemistries, but ONT R9 results deviated slightly more from the Illumina results. ONT R10 results showed trends very similar to Illumina, and we found that integrating data sets sequenced by either Illumina or ONT R10 for different isolates into the same analysis produced stable and highly accurate phylogenomic results. The resulting phylogenies for these two outbreaks stabilized after ~20 hours of sequencing for ONT R9 and ~8 hours for ONT R10. This study provides a proof of concept for using ONT R10, either in isolation or in combination with Illumina, for rapid and accurate bacterial SNP-based outbreak investigation.

M3 - 10.1128/jcm.01576-23 ER - TY - JOUR T1 - Development of a reverse transcriptase digital droplet polymerase chain reaction‐based approach for SARS‐CoV‐2 variant surveillance in wastewater JF - Water Environment Research Y1 - 2024 A1 - Laura Van Poelvoorde A1 - Andrea Gobbo A1 - S. Nauwelaerts A1 - Bavo Verhaegen A1 - Marie Lesenfants A1 - Raphael Janssens A1 - Veronik Hutse A1 - Marie-Alice Fraiture A1 - Sigrid C.J. De Keersmaecker A1 - Philippe Herman A1 - Koenraad Van Hoorde A1 - Nancy Roosens VL - 96 CP - 3 M3 - 10.1002/wer.10999 ER - TY - JOUR T1 - Pilot market surveillance of GMM contaminations in alpha-amylase food enzyme products: A detection strategy strengthened by a newly developed qPCR method targeting a GM Bacillus licheniformis producing alpha-amylase JF - Food Chemistry: Molecular Sciences Y1 - 2024 A1 - Marie-Alice Fraiture A1 - Andrea Gobbo A1 - Chloé Guillitte A1 - Ugo Marchesi A1 - Daniela Verginelli A1 - Joke De Greve A1 - Jolien D'aes A1 - Kevin Vanneste A1 - N. Papazova A1 - Nancy Roosens VL - 8 M3 - 10.1016/j.fochms.2023.100186 ER - TY - RPRT T1 - Wastewater-based epidemiological surveillance - Methodological appendix Y1 - 2024 A1 - Raphael Janssens A1 - Hadrien Maloux A1 - Sven Hanoteaux A1 - Laura Van Poelvoorde A1 - Nancy Roosens A1 - Bavo Verhaegen A1 - Julie Linussio A1 - Koenraad Van Hoorde A1 - Steven Van Gucht A1 - Karin De Ridder A1 - Koen Blot A1 - Veronik Hutse A1 - Marie Lesenfants KW - applied genomics KW - environment KW - epidemiology KW - Surveillance KW - wastewater KW - wastewater surveillance KW - wastewater-based epidemiology PB - Sciensano CY - Brussels, Belgium UR - https://www.sciensano.be/en/biblio/wastewater-based-epidemiological-surveillance-methodological-appendix ER - TY - RPRT T1 - Wastewater-based epidemiological surveillance - Weekly report Y1 - 2024 A1 - Raphael Janssens A1 - Hadrien Maloux A1 - Sven Hanoteaux A1 - Laura Van Poelvoorde A1 - Nancy Roosens A1 - Bavo Verhaegen A1 - Julie Linussio A1 - Koenraad Van Hoorde A1 - Steven Van Gucht A1 - Karin De Ridder A1 - Koen Blot A1 - Veronik Hutse A1 - Marie Lesenfants KW - applied genomics KW - environment KW - epidemiology KW - Surveillance KW - wastewater KW - wastewater surveillance KW - wastewater-based epidemiology PB - Sciensano CY - Brussels, Belgium UR - https://www.sciensano.be/en/biblio/wastewater-based-epidemiological-surveillance-weekly-report ER - TY - JOUR T1 - Comparison of 6 DNA extraction methods for isolation of high yield of high molecular weight DNA suitable for shotgun metagenomics Nanopore sequencing to detect bacteriaAbstractBackgroundResultsConclusion JF - BMC Genomics Y1 - 2023 A1 - Mathieu Gand A1 - Bram Bloemen A1 - Kevin Vanneste A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - DNA extraction KW - HMW DNA KW - Long-read KW - Metagenomics KW - Method evaluation KW - nanopore AB -

Background: Oxford Nanopore Technologies (ONT) offers an accessible platform for long-read sequencing, which improves the reconstruction of genomes and helps to resolve complex genomic contexts, especially in the case of metagenome analysis. To take the best advantage of long-read sequencing, DNA extraction methods must be able to isolate pure high molecular weight (HMW) DNA from complex metagenomics samples, without introducing any
bias. New methods released on the market, and protocols developed at the research level, were specifically designed for this application and need to be assessed.
Results: In this study, with different bacterial cocktail mixes, analyzed as pure or spiked in a synthetic fecal matrix, we evaluated the performances of 6 DNA extraction methods using various cells lysis and purification techniques, from quick and easy, to more time-consuming and gentle protocols, including a portable method for on-site application. In addition to the comparison of the quality, quantity and purity of the extracted DNA, the performance
obtained when doing Nanopore sequencing on a MinION flow cell was also tested. From the obtained results, the Quick-DNA HMW MagBead Kit (Zymo Research) was selected as producing the best yield of pure HMW DNA. Furthermore, this kit allowed an accurate detection, by Nanopore sequencing, of almost all the bacterial species present in a complex mock community.
Conclusion: Amongst the 6 tested methods, the Quick-DNA HMW MagBead Kit (Zymo Research) was considered as the most suitable for Nanopore sequencing and would be recommended for bacterial metagenomics studies using this technology

VL - 24 CP - 1 M3 - 10.1186/s12864-023-09537-5 ER - TY - Generic T1 - Contaminations of AMR genes linked to the presence of genetically modified microorganisms in the food and feed chain Y1 - 2023 A1 - Marie-Alice Fraiture A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens PB - Antimicrobial resistance: a challenge for public health, animal health and environment, AMCRA CY - Brussels, Belgium ER - TY - Generic T1 - Detection of food and feed plant products obtained by targeted mutagenesis and cisgenesis Y1 - 2023 A1 - A. Barbante A1 - W. Broothaerts A1 - M. Burns A1 - F. Debode A1 - M. De Giacomo A1 - M. De Loose A1 - T. Demšar A1 - K. Eckermann A1 - Marie-Alice Fraiture A1 - L. Grantina-Ievina A1 - P. Gürtler A1 - J. Mallet A1 - M. Mazzara A1 - E. Perri A1 - T. Prins A1 - Nancy Roosens A1 - C. Savini A1 - S. Trapmann A1 - C. Weidner A1 - X. Zaoui JF - Publications Office of the European Union PB - European Network of GMO Laboratories CY - Luxembourg VL - JRC133689 M3 - https://doi.org/10.2760/007925 ER - TY - JOUR T1 - Development of a Droplet Digital PCR to Monitor SARS-CoV-2 Omicron Variant BA.2 in Wastewater Samples JF - Microorganisms Y1 - 2023 A1 - Laura Van Poelvoorde A1 - Corinne Picalausa A1 - Andrea Gobbo A1 - Bavo Verhaegen A1 - Marie Lesenfants A1 - Philippe Herman A1 - Koenraad Van Hoorde A1 - Nancy Roosens KW - SARS-CoV-2; omicron; VOC; mutation; RT-ddPCR; variant detection; wastewater surveillance AB -

Wastewater-based surveillance can be used as a complementary method to other SARS-CoV-2 surveillance systems. It allows the emergence and spread of infections and SARS-CoV-2 variants to be monitored in time and place. This study presents an RT-ddPCR method that targets the T19I amino acid mutation in the spike protein of the SARS-CoV-2 genomes, which is specific to the BA.2 variant (omicron). The T19I assay was evaluated both in silico and in vitro for its inclusivity, sensitivity, and specificity. Moreover, wastewater samples were used as a proof of concept to monitor and quantify the emergence of the BA.2 variant from January until May 2022 in the Brussels-Capital Region which covers a population of more than 1.2 million inhabitants. The in silico analysis showed that more than 99% of the BA.2 genomes could be characterized using the T19I assay. Subsequently, the sensitivity and specificity of the T19I assay were successfully experimentally evaluated. Thanks to our specific method design, the positive signal from the mutant probe and wild-type probe of the T19I assay was measured and the proportion of genomes with the T19I mutation, characteristic of the BA.2 mutant, compared to the entire SARS-CoV-2 population was calculated. The applicability of the proposed RT-ddPCR method was evaluated to monitor and quantify the emergence of the BA.2 variant over time. To validate this assay as a proof of concept, the measurement of the proportion of a specific circulating variant with genomes containing the T19I mutation in comparison to the total viral population was carried out in wastewater samples from wastewater treatment plants in the Brussels-Capital Region in the winter and spring of 2022. This emergence and proportional increase in BA.2 genomes correspond to what was observed in the surveillance using respiratory samples; however, the emergence was observed slightly earlier, which suggests that wastewater sampling could be an early warning system and could be an interesting alternative to extensive human testing.

VL - 11 CP - 3 M3 - https://doi.org/10.3390/microorganisms11030729 ER - TY - JOUR T1 - Development of a portable on-site applicable metagenomic data generation workflow for enhanced pathogen and antimicrobial resistance surveillanceAbstract JF - Scientific Reports Y1 - 2023 A1 - Bram Bloemen A1 - Mathieu Gand A1 - Kevin Vanneste A1 - Marchal, Kathleen A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker VL - 13 CP - 1 M3 - 10.1038/s41598-023-46771-z ER - TY - JOUR T1 - Development of a taxon-specific real-time polymerase chain reaction method to detect Trichoderma reesei contaminations in fermentation products. Fermentation, 9 (11), 926 JF - Fermentation Y1 - 2023 A1 - Marie-Alice Fraiture A1 - Andrea Gobbo A1 - N. Papazova A1 - Nancy Roosens KW - food control; fungal fermentation products; real-time PCR; detection; Trichoderma reesei AB -

Recently, a genetically modified microorganism (GMM) detection strategy using real-time PCR technology was developed to control fermentation products commercialized in the food and feed chain, allowing several unexpected GMM contaminations to be highlighted. Currently, only bacterial strains are targeted by this strategy. Given that fungal strains, like Trichoderma reesei, are also frequently used by the food industry to produce fermentation products, a novel real-time PCR method specific to this fungal species was developed and validated in this study to reinforce the GMM detection strategy. Designed to cover a sequence of 130 bp from the translation elongation factor alpha 1 (Tef1) gene of T. reesei, this real-time PCR method, namely TR, allows for the screening of commercial fermentation products contaminated with T. reesei, genetically modified or not, which is one of the major fungal species used as an industrial platform for the manufacturing of fermentation products. The developed real-time PCR TR method was assessed as specific and sensitive (LOD95% = eight copies). In addition, the developed real-time PCR TR method performance was confirmed to be in line with the “Minimum Performance Requirements for Analytical Methods of GMO Testing” of the European Network of GMO Laboratories. The validated real-time PCR TR method was also demonstrated to be applicable to commercial microbial fermentation products. Based on all these results, the novel real-time PCR TR method was assessed as valuable for strengthening the current GMM detection strategy regarding major fungal species used by the food industry to produce microbial fermentation products.

VL - 9 M3 - https://doi.org/10.3390/fermentation9110926 ER - TY - JOUR T1 - Development of a Taxon-Specific Real-Time Polymerase Chain Reaction Method to Detect Trichoderma reesei Contaminations in Fermentation Products JF - Fermentation Y1 - 2023 A1 - Marie-Alice Fraiture A1 - Andrea Gobbo A1 - N. Papazova A1 - Nancy Roosens VL - 9 CP - 11 M3 - 10.3390/fermentation9110926 ER - TY - JOUR T1 - Development of a Taxon-Specific Real-Time Polymerase Chain Reaction Method to Detect Trichoderma reesei Contaminations in Fermentation Products JF - Fermentation Y1 - 2023 A1 - Marie-Alice Fraiture A1 - Andrea Gobbo A1 - N. Papazova A1 - Nancy Roosens AB -

Recently, a genetically modified microorganism (GMM) detection strategy using real-time
PCR technology was developed to control fermentation products commercialized in the food and feed
chain, allowing several unexpected GMM contaminations to be highlighted. Currently, only bacterial
strains are targeted by this strategy. Given that fungal strains, like Trichoderma reesei, are also frequently
used by the food industry to produce fermentation products, a novel real-time PCR method specific to
this fungal species was developed and validated in this study to reinforce the GMM detection strategy.
Designed to cover a sequence of 130 bp from the translation elongation factor alpha 1 (Tef1) gene of
T. reesei, this real-time PCR method, namely TR, allows for the screening of commercial fermentation
products contaminated with T. reesei, genetically modified or not, which is one of the major fungal
species used as an industrial platform for the manufacturing of fermentation products. The developed
real-time PCR TR method was assessed as specific and sensitive (LOD95% = eight copies). In addition,
the developed real-time PCR TR method performance was confirmed to be in line with the “Minimum
Performance Requirements for Analytical Methods of GMO Testing” of the European Network of
GMO Laboratories. The validated real-time PCR TR method was also demonstrated to be applicable
to commercial microbial fermentation products. Based on all these results, the novel real-time PCR
TR method was assessed as valuable for strengthening the current GMM detection strategy regarding
major fungal species used by the food industry to produce microbial fermentation products.

VL - 9 CP - 11 M3 - 10.3390/fermentation9110926 ER - TY - JOUR T1 - Development of Digital Droplet PCR Targeting the Influenza H3N2 Oseltamivir-Resistant E119V Mutation and Its Performance through the Use of Reverse Genetics Mutants JF - Current Issues in Molecular Biology Y1 - 2023 A1 - Laura Van Poelvoorde A1 - François Dufrasne A1 - Steven Van Gucht A1 - Xavier Saelens A1 - Nancy Roosens VL - 45 CP - 3 M3 - 10.3390/cimb45030165 ER - TY - JOUR T1 - Development of Digital Droplet PCR Targeting the Influenza H3N2 Oseltamivir-Resistant E119V Mutation and Its Performance through the Use of Reverse Genetics Mutants. JF - Curr Issues Mol Biol Y1 - 2023 A1 - Laura Van Poelvoorde A1 - François Dufrasne A1 - Steven Van Gucht A1 - Saelens, Xavier A1 - Nancy Roosens KW - INFLUENZA KW - Oseltamivir KW - PCR KW - resistance AB -

The monitoring of antiviral-resistant influenza virus strains is important for public health given the availability and use of neuraminidase inhibitors and other antivirals to treat infected patients. Naturally occurring oseltamivir-resistant seasonal H3N2 influenza virus strains often carry a glutamate-to-valine substitution at position 119 in the neuraminidase (E119V-NA). Early detection of resistant influenza viruses is important for patient management and for the rapid containment of antiviral resistance. The neuraminidase inhibition assay allows the phenotypical identification of resistant strains; however, this test often has limited sensitivity with high variability depending on the virus strain, drugs and assays. Once a mutation such as E119V-NA is known, highly sensitive PCR-based genotypic assays can be used to identify the prevalence of such mutant influenza viruses in clinical samples. In this study, based on an existing reverse transcriptase real-time PCR (RT-qPCR) assay, we developed a reverse transcriptase droplet digital PCR assay (RT-ddPCR) to detect and quantify the frequency of the E119V-NA mutation. Furthermore, reverse genetics viruses carrying this mutation were created to test the performance of the RT-ddPCR assay and compare it to the standard phenotypic NA assay. We also discuss the advantage of using an RT-ddPCR instead of qPCR method in the context of viral diagnostics and surveillance.

VL - 45 CP - 3 M3 - 10.3390/cimb45030165 ER - TY - Generic T1 - A new open strategy for impurity surveillance of commercial microbial fermentation food products Y1 - 2023 A1 - Marie-Alice Fraiture A1 - Nancy Roosens A1 - N. Papazova A1 - Sigrid C.J. De Keersmaecker A1 - Kevin Vanneste A1 - Rik Orval A1 - Yari Van Laere A1 - Frédéric Debode A1 - Marc De Loose A1 - Christof Van Poucke A1 - Nathalie Gillard PB - 27th Conference on Food Microbiology - BSFM CY - Brussels, Belgium ER - TY - Generic T1 - Novel threats- State of the art of synthetic biology Y1 - 2023 A1 - T. Bjordal Johansen A1 - E.H. Madslien A1 - M. Forsman A1 - F. Ekström A1 - E.M. Fykse A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Kevin Vanneste A1 - Marie-Alice Fraiture A1 - B. Palyi A1 - M. Knepr-Segina A1 - L. Zmak A1 - I. Tabain A1 - H. Janković A1 - J. Jones PB - Medical Biodefense Conference 2023 - MBDC CY - Munich, Germany ER - TY - JOUR T1 - Retrospective surveillance of viable Bacillus cereus group contaminations in commercial food and feed vitamin B2 products sold on the Belgian market using whole-genome sequencing JF - Frontiers in Microbiology Y1 - 2023 A1 - Bert Bogaerts A1 - Marie-Alice Fraiture A1 - Tom Van Nieuwenhuysen A1 - Bram Jacobs A1 - Koenraad Van Hoorde A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Kevin Vanneste KW - Bacillus cereus KW - Food Additives KW - riboflavin KW - Surveillance KW - whole-genome sequencing AB -

Bacillus cereus is a spore-forming bacterium that occurs as a contaminant in food and feed, occasionally resulting in food poisoning through the production of various toxins. In this study, we retrospectively characterized viable B. cereus sensu lato (s.l.) isolates originating from commercial vitamin B2 feed and food additives collected between 2016 and 2022 by the Belgian Federal Agency for the Safety of the Food Chain from products sold on the Belgian market. In total, 75 collected product samples were cultured on a general medium and, in case of bacterial growth, two isolates per product sample were collected and characterized using whole-genome sequencing (WGS) and subsequently characterized in terms of sequence type (ST), virulence gene profile, antimicrobial resistance (AMR) gene profile, plasmid content, and phylogenomic relationships. Viable B. cereus was identified in 18 of the 75 (24%) tested products, resulting in 36 WGS datasets, which were classified into eleven different STs, with ST165 (n = 10) and ST32 (n = 8) being the most common. All isolates carried multiple genes encoding virulence factors, including cytotoxin K-2 (52.78%) and cereulide (22.22%). Most isolates were predicted to be resistant to beta-lactam antibiotics (100%) and fosfomycin (88.89%), and a subset was predicted to be resistant to streptothricin (30.56%). Phylogenomic analysis revealed that some isolates obtained from different products were closely related or even identical indicating a likely common origin, whereas for some products the two isolates obtained did not show any close relationship to each other or other isolates found in other products. This study reveals that potentially pathogenic and drug-resistant B. cereus s.l. can be present in food and feed vitamin B2 additives that are commercially available, and that more research is warranted to assess whether their presence in these types of products poses a threat to consumers.

VL - 14 M3 - 10.3389/fmicb.2023.1173594 ER - TY - Generic T1 - Strain-level characterization without culture enrichment? Easing and accelerating outbreak investigation using shotgun metagenomics facilitated with nanopore adaptive sampling Y1 - 2023 A1 - Florence E Buytaers A1 - Bavo Verhaegen A1 - Tom Van Nieuwenhuysen A1 - Nancy Roosens A1 - Kevin Vanneste A1 - Kathleen Marchal A1 - Sigrid C.J. De Keersmaecker KW - adaptive sampling KW - nanopore KW - outbreak investigation KW - shotgun metagenomics AB -

Introduction

Traditionally, the investigation of suspect food samples during an outbreak investigation requires the samples to be enriched in order to reach detectable levels. However, a bias can occur during the enrichment of the food due to competition between the various living organisms or the cultivation parameters. Moreover, the current methods require a subsequent but not always successful isolation. In part due to these two drawbacks, not all outbreaks can be resolved. Shotgun metagenomics is a sequencing method involving the analysis of the genetic material directly extracted from a sample, without the need for an isolation of the bacteria. By sequencing and analysing the DNA of the sample, it can identify the presence of various pathogens to the strain level. Moreover, adaptive sampling is a new tool proposed during nanopore sequencing to target certain DNA strands to be sequenced preferably. The combination of metagenomics and adaptive sampling could allow to circumvent the isolation but also the enrichment of the food sample.

Materials and Methods

As a proof-of-concept, we explored the use of adaptive sampling using various databases (depletion of the food matrix i.e. potato, enrichment of a selection of foodborne pathogens, enrichment of a database of Staphylococcus aureus) compared to shotgun metagenomics without adaptive sampling on DNA of mashed potatoes spiked with DNA of S. aureus at a level of 0.5%. The strain was previously associated with a foodborne outbreak. Additionally, the living S. aureus strain was spiked into mashed potatoes at a level of 105 CFU per 25 grams and three DNA extraction kits were tested, in combination with two databases for adaptive sampling, following whole genome amplification to increase the amount of genetic material to meet sequencing standards. The strain-level data analysis was performed as previously described (Buytaers et al. 2021).

Discussion

Our results showed that the combination of whole genome amplification, metagenomics and adaptive sampling using a database of S. aureus genomes outperformed shotgun metagenomics and adaptive sampling using other databases. It allowed strain-level analysis of foodborne outbreaks without the need for culture enrichment, thereby enabling faster investigations and facilitating precise pathogen characterization, contributing to improved food safety and public health.

JF - BSFM PB - BSFM CY - Brussels, Belgium ER - TY - JOUR T1 - Targeted High-Throughput Sequencing Enables the Detection of Single Nucleotide Variations in CRISPR/Cas9 Gene-Edited Organisms JF - Foods Y1 - 2023 A1 - Marie-Alice Fraiture A1 - Jolien D'aes A1 - Emmanuel Guiderdoni A1 - Anne-Cécile Meunier A1 - Thomas Delcourt A1 - Stefan Hoffman A1 - Els Vandermassen A1 - Sigrid C.J. De Keersmaecker A1 - Kevin Vanneste A1 - Nancy Roosens KW - CRISPR/Cas9 KW - detection KW - food and feed chain KW - Genetically modified organism KW - high-throughput sequencing KW - PCR-based enrichment KW - single nucleotide variations KW - Transgenic Rice AB -

Similar to genetically modified organisms (GMOs) produced by classical genetic engineering, gene-edited (GE) organisms and their derived food/feed products commercialized on the European Union market fall within the scope of European Union Directive 2001/18/EC. Consequently, their control in the food/feed chain by GMO enforcement laboratories is required by the competent authorities to guarantee food/feed safety and traceability (2003/1829/EC; 2003/1830/EC). However, their detection is potentially challenging at both the analytical and interpretation levels since this requires methodological approaches that can target and detect a specific single nucleotide variation (SNV) introduced into a GE organism. In this study, we propose a targeted high-throughput sequencing approach, including (i) a prior PCR-based enrichment step to amplify regions of interest, (ii) a sequencing step, and (iii) a data analysis methodology to identify SNVs of interest. To investigate if the performance of this targeted high-throughput sequencing approach is compatible with the performance criteria used in the GMO detection field, several samples containing different percentages of a GE rice line carrying a single adenosine insertion in OsMADS26 were prepared and analyzed. The SNV of interest in samples containing the GE rice line could successfully be detected, both at high and low percentages. No impact related to food processing or to the presence of other crop species was observed. The present proof-of-concept study has allowed us to deliver the first experimental-based evidence indicating that the proposed targeted high-throughput sequencing approach may constitute, in the future, a specific and sensitive tool to support the safety and traceability of the food/feed chain regarding GE plants carrying SNVs.

VL - 12 CP - 3 M3 - 10.3390/foods12030455 ER - TY - JOUR T1 - Time series modelling for wastewater-based epidemiology of COVID-19: A nationwide study in 40 wastewater treatment plants of Belgium, February 2021 to June 2022 JF - Science of The Total Environment Y1 - 2023 A1 - Xander Bertels A1 - Sven Hanoteaux A1 - Raphael Janssens A1 - Hadrien Maloux A1 - Bavo Verhaegen A1 - Peter Delputte A1 - Tim Boogaerts A1 - Alexander L.N. van Nuijs A1 - Delphine Brogna A1 - Catherine Linard A1 - Marescaux, Jonathan A1 - Christian Didy A1 - Rosalie Pype A1 - Nancy Roosens A1 - Koenraad Van Hoorde A1 - Marie Lesenfants A1 - Lies Lahousse VL - 899 M3 - 10.1016/j.scitotenv.2023.165603 ER - TY - JOUR T1 - Time series modelling for wastewater-based epidemiology of COVID-19: A nationwide study in 40 wastewater treatment plants of Belgium, February 2021 to June 2022 JF - Science of The Total Environment Y1 - 2023 A1 - Xander Bertels A1 - Sven Hanoteaux A1 - Raphael Janssens A1 - Hadrien Maloux A1 - Bavo Verhaegen A1 - Peter Delputte A1 - Tim Boogaerts A1 - Alexander L.N. van Nuijs A1 - Delphine Brogna A1 - Catherine Linard A1 - Marescaux, Jonathan A1 - Christian Didy A1 - Rosalie Pype A1 - Nancy Roosens A1 - Koenraad Van Hoorde A1 - Marie Lesenfants A1 - Lies Lahousse KW - ARIMA KW - COVID-19 KW - Flow rate KW - PMMoV KW - wastewater surveillance AB -

Background: Wastewater-based epidemiology (WBE) has been implemented to monitor surges of COVID-19. Yet,
multiple factors impede the usefulness of WBE and quantitative adjustment may be required.
Aim: We aimed to model the relationship between WBE data and incident COVID-19 cases, while adjusting for
confounders and autocorrelation.
Methods: This nationwide WBE study includes data from 40 wastewater treatment plants (WWTPs) in Belgium
(02/2021–06/2022). We applied ARIMA-based modelling to assess the effect of daily flow rate, pepper mild
mottle virus (PMMoV) concentration, a measure of human faeces in wastewater, and variants (alpha, delta, and
omicron strains) on SARS-CoV-2 RNA levels in wastewater. Secondly, adjusted WBE metrics at different lag times
were used to predict incident COVID-19 cases. Model selection was based on AICc minimization.
Results: In 33/40 WWTPs, RNA levels were best explained by incident cases, flow rate, and PMMoV. Flow rate
and PMMoV were associated with -13.0 % (95 % prediction interval: -26.1 to +0.2 %) and +13.0 % (95 %
prediction interval: +5.1 to +21.0 %) change in RNA levels per SD increase, respectively. In 38/40 WWTPs,
variants did not explain variability in RNA levels independent of cases. Furthermore, our study shows that RNA
levels can lead incident cases by at least one week in 15/40 WWTPs. The median population size of leading
WWTPs was 85.1 % larger than that of non‑leading WWTPs. In 17/40 WWTPs, however, RNA levels did not lead
or explain incident cases in addition to autocorrelation.
Conclusion: This study provides quantitative insights into key determinants of WBE, including the effects of
wastewater flow rate, PMMoV, and variants. Substantial inter-WWTP variability was observed in terms of
explaining incident cases. These findings are of practical importance to WBE practitioners and show that the
early-warning potential of WBE is WWTP-specific and needs validation.

VL - 899 M3 - 10.1016/j.scitotenv.2023.165603 ER - TY - JOUR T1 - Transforming Shiga toxin-producing surveillance through whole genome sequencing in food safety practices. JF - Front Microbiol Y1 - 2023 A1 - Stéphanie Nouws A1 - Bavo Verhaegen A1 - Sarah Denayer A1 - Florence Crombé A1 - Denis Piérard A1 - Bert Bogaerts A1 - Kevin Vanneste A1 - Kathleen Marchal A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - Food Safety KW - implementation KW - Shiga toxin-producing Escherichia coli KW - Surveillance KW - whole genome sequencing AB -

INTRODUCTION: Shiga toxin-producing (STEC) is a gastrointestinal pathogen causing foodborne outbreaks. Whole Genome Sequencing (WGS) in STEC surveillance holds promise in outbreak prevention and confinement, in broadening STEC epidemiology and in contributing to risk assessment and source attribution. However, despite international recommendations, WGS is often restricted to assist outbreak investigation and is not yet fully implemented in food safety surveillance across all European countries, in contrast to for example in the United States.

METHODS: In this study, WGS was retrospectively applied to isolates collected within the context of Belgian food safety surveillance and combined with data from clinical isolates to evaluate its benefits. A cross-sector WGS-based collection of 754 strains from 1998 to 2020 was analyzed.

RESULTS: We confirmed that WGS in food safety surveillance allows accurate detection of genomic relationships between human cases and strains isolated from food samples, including those dispersed over time and geographical locations. Identifying these links can reveal new insights into outbreaks and direct epidemiological investigations to facilitate outbreak management. Complete WGS-based isolate characterization enabled expanding epidemiological insights related to circulating serotypes, virulence genes and antimicrobial resistance across different reservoirs. Moreover, associations between virulence genes and severe disease were determined by incorporating human metadata into the data analysis. Gaps in the surveillance system were identified and suggestions for optimization related to sample centralization, harmonizing isolation methods, and expanding sampling strategies were formulated.

DISCUSSION: This study contributes to developing a representative WGS-based collection of circulating STEC strains and by illustrating its benefits, it aims to incite policymakers to support WGS uptake in food safety surveillance.

VL - 14 M3 - 10.3389/fmicb.2023.1204630 ER - TY - JOUR T1 - Using a combination of short and long read sequencing to investigate the diversity in plasmid- and chromosomally encoded extended-spectrum-beta-lactamases (ESBL) in clinical Shigella and Salmonella isolates in Belgium JF - Microbial Genomics Y1 - 2023 A1 - Bas Berbers A1 - Kevin Vanneste A1 - Nancy Roosens A1 - Marchal,K. A1 - Pieter-Jan Ceyssens A1 - Sigrid C.J. De Keersmaecker KW - Antimicrobial resistance KW - extended spectrum beta-lactamase KW - Plasmids KW - Salmonella KW - Shigella KW - whole genome sequencing. VL - 9:000925 M3 - DOI 10.1099/mgen.0.000925 ER - TY - JOUR T1 - Using a combination of short- and long-read sequencing to investigate the diversity in plasmid- and chromosomally encoded extended-spectrum beta-lactamases (ESBLs) in clinical Shigella and Salmonella isolates in Belgium JF - Microbial Genomics Y1 - 2023 A1 - Bas Berbers A1 - Kevin Vanneste A1 - Nancy Roosens A1 - Marchal, Kathleen A1 - Pieter-Jan Ceyssens A1 - Sigrid C.J. De Keersmaecker VL - 9 CP - 1 M3 - 10.1099/mgen.0.000925 ER - TY - Generic T1 - Whole-genome sequencing-based phylogenomic investigation of unexpected Bacillus cereus contaminations collected from commercial vitamin B2 food and feed additives sold on the Belgian market Y1 - 2023 A1 - Bert Bogaerts A1 - Marie-Alice Fraiture A1 - Astrid, Huwaert A1 - Tom Van Nieuwenhuysen A1 - Bram Jacobs A1 - Koenraad Van Hoorde A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Kevin Vanneste JF - Applied Bioinformatics and Public Health Microbiology PB - Wellcome Genome Campus CY - Hinxton, OK ER - TY - JOUR T1 - Whole-Genome Sequencing-Based Screening of MRSA in Patients and Healthcare Workers in Public Hospitals in Benin. JF - Microorganisms Y1 - 2023 A1 - Laurence Carine Yehouenou A1 - Bert Bogaerts A1 - Kevin Vanneste A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Arsène A Kpangon A1 - Affolabi, Dissou A1 - Simon, Anne A1 - Francis Moise Dossou A1 - Olivia Dalleur KW - HEALTHCARE WORKERS KW - low- or middle-income countries KW - Methicillin-Resistant Staphylococcus aureus KW - whole-genome sequencing. AB -

Methicillin-resistant (MRSA) constitutes a serious public health concern, with a considerable impact on patients' health, and substantial healthcare costs. In this study, patients and healthcare workers (HCWs) from six public hospitals in Benin were screened for MRSA. Strains were identified as MRSA using conventional microbiological methods in Benin, and confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in Belgium. Whole-genome sequencing (WGS) was used on the confirmed MRSA isolates, to characterize their genomic content and study their relatedness. Amongst the 305 isolates (304 wound swabs and 61 nasal swabs) that were collected from patients and HCWs, we detected 32 and 15 cases of MRSA, respectively. From this collection, 27 high-quality WGS datasets were obtained, which carried numerous genes and mutations associated with antimicrobial resistance. The gene was detected in all the sequenced isolates. These isolates were assigned to five sequence types (STs), with ST8 (55.56%, n = 15/27), ST152 (18.52%, n = 5/27), and ST121 (18.52%, n = 5/27) being the most common. These 27 isolates carried multiple virulence genes, including the genes encoding the Panton-Valentine leukocidin toxin (48.15%, n = 13/27), and the gene (29.63%, n = 8/27), associated with toxic shock syndrome. This study highlights the need to implement a multimodal strategy for reducing the risk of the cross-transmission of MRSA in hospitals.

VL - 11 CP - 8 M3 - 10.3390/microorganisms11081954 ER - TY - JOUR T1 - Assessment of the Feasibility of a Future Integrated Larger-Scale Epidemiological Study to Evaluate Health Risks of Air Pollution Episodes in Children. JF - Int J Environ Res Public Health Y1 - 2022 A1 - S. Nauwelaerts A1 - Koen De Cremer A1 - Natalia Bustos Sierra A1 - Mathieu Gand A1 - D. Van Geel A1 - Delvoye, Maud A1 - Els Vandermassen A1 - Jordy Vercauteren A1 - Christophe Stroobants A1 - Bernard, Alfred A1 - Nelly D Saenen A1 - Nawrot, Tim S A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - Air Pollutants KW - air pollution KW - Biomarkers KW - Child KW - environmental exposure KW - Epidemiologic Studies KW - Feasibility Studies KW - Humans KW - Particulate Matter AB -

Air pollution exposure can lead to exacerbation of respiratory disorders in children. Using sensitive biomarkers helps to assess the impact of air pollution on children's respiratory health and combining protein, genetic and epigenetic biomarkers gives insights on their interrelatedness. Most studies do not contain such an integrated approach and investigate these biomarkers individually in blood, although its collection in children is challenging. Our study aimed at assessing the feasibility of conducting future integrated larger-scale studies evaluating respiratory health risks of air pollution episodes in children, based on a qualitative analysis of the technical and logistic aspects of a small-scale field study involving 42 children. This included the preparation, collection and storage of non-invasive samples (urine, saliva), the measurement of general and respiratory health parameters and the measurement of specific biomarkers (genetic, protein, epigenetic) of respiratory health and air pollution exposure. Bottlenecks were identified and modifications were proposed to expand this integrated study to a higher number of children, time points and locations. This would allow for non-invasive assessment of the impact of air pollution exposure on the respiratory health of children in future larger-scale studies, which is critical for the development of policies or measures at the population level.

VL - 19 CP - 14 M3 - 10.3390/ijerph19148531 ER - TY - JOUR T1 - Detection and identification of authorized and unauthorized GMOs using high-throughput sequencing with the support of a sequence-based GMO database JF - Food Chemistry: Molecular Sciences Y1 - 2022 A1 - Assia Saltykova A1 - Julien Van Braekel A1 - N. Papazova A1 - Marie-Alice Fraiture A1 - Deforce, Dieter A1 - Kevin Vanneste A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens VL - 4 M3 - 10.1016/j.fochms.2022.100096 ER - TY - JOUR T1 - Development of a taxon-specific real-time PCR method targeting the Bacillus subtilis group to strengthen the control of genetically modified bacteria in fermentation products JF - Fermentation Y1 - 2022 A1 - Marie-Alice Fraiture A1 - Andrea Gobbo A1 - N. Papazova A1 - Nancy Roosens KW - additives and flavorings KW - Bacillus subtilis group KW - Enzymes KW - food and feed safety concerns KW - real-time PCR detection KW - Unauthorized genetically modified microorganisms AB -

Most of the bacteria that are used to produce fermentation products, such as enzymes, additives and flavorings, belong to the Bacillus subtilis group. Recently, unexpected contaminations with unauthorized genetically modified (GM) bacteria (viable cells and associated DNA) that were carrying antimicrobial resistance (AMR) genes was noticed in several microbial fermentation products that have been commercialized on the food and feed market. These contaminations consisted of GM Bacillus species belonging to the B. subtilis group. In order to screen for the potential presence of such contaminations, in this study we have developed a new real-time PCR method targeting the B. subtilis group, including B. subtilisB. licheniformisB. amyloliquefaciens and B. velezensis. The method’s performance was successfully assessed as specific and sensitive, complying with the Minimum Performance Requirements for Analytical Methods of GMO Testing that is used as a standard by the GMO enforcement laboratories. The method’s applicability was also tested on 25 commercial microbial fermentation products. In addition, this method was developed to be compatible with the PCR-based strategy that was recently developed for the detection of unauthorized GM bacteria. This taxon-specific method allows the strengthening of the set of screening markers that are targeting key sequences that are frequently found in GM bacteria (AMR genes and shuttle vector), reinforcing control over the food and feed chain in order to guarantee its safety and traceability.

VL - 8 CP - 2 M3 - https://doi.org/10.3390/fermentation8020078 ER - TY - JOUR T1 - A general approach to identify low-frequency variants within influenza samples collected during routine surveillance. JF - Microb Genom Y1 - 2022 A1 - Laura Van Poelvoorde A1 - Thomas Delcourt A1 - Marnik Vuylsteke A1 - Sigrid C.J. De Keersmaecker A1 - Isabelle Thomas A1 - Steven Van Gucht A1 - Saelens, Xavier A1 - Nancy Roosens A1 - Kevin Vanneste KW - COVID-19 KW - Genome, Viral KW - Humans KW - Influenza A Virus, H3N2 Subtype KW - Influenza, Human KW - SARS-CoV-2 AB -

Influenza viruses exhibit considerable diversity between hosts. Additionally, different quasispecies can be found within the same host. High-throughput sequencing technologies can be used to sequence a patient-derived virus population at sufficient depths to identify low-frequency variants (LFV) present in a quasispecies, but many challenges remain for reliable LFV detection because of experimental errors introduced during sample preparation and sequencing. High genomic copy numbers and extensive sequencing depths are required to differentiate false positive from real LFV, especially at low allelic frequencies (AFs). This study proposes a general approach for identifying LFV in patient-derived samples obtained during routine surveillance. Firstly, validated thresholds were determined for LFV detection, whilst balancing both the cost and feasibility of reliable LFV detection in clinical samples. Using a genetically well-defined population of influenza A viruses, thresholds of at least 10 genomes per microlitre and AF of ≥5 % were established as detection limits. Secondly, a subset of 59 retained influenza A (H3N2) samples from the 2016-2017 Belgian influenza season was composed. Thirdly, as a proof of concept for the added value of LFV for routine influenza monitoring, potential associations between patient data and whole genome sequencing data were investigated. A significant association was found between a high prevalence of LFV and disease severity. This study provides a general methodology for influenza LFV detection, which can also be adopted by other national influenza reference centres and for other viruses such as SARS-CoV-2. Additionally, this study suggests that the current relevance of LFV for routine influenza surveillance programmes might be undervalued.

VL - 8 CP - 9 M3 - 10.1099/mgen.0.000867 ER - TY - Generic T1 - Introduction of WGS in food microbiology: advantages and challenges Y1 - 2022 A1 - Bavo Verhaegen A1 - N Botteldoorn A1 - Vera Cantaert A1 - de Zutter, Lieven A1 - Véronique Delcenserie A1 - Annemie Geeraerd A1 - Mahillon, Jacques A1 - Marcella Mori A1 - Pochet, Brigitte A1 - Nancy Roosens A1 - Koenraad Van Hoorde A1 - Kim Feys A1 - Herman, Lieve KW - antimicrobial resistance (AMR) KW - Food Safety KW - foodborne outbreak investigation KW - Monitoring KW - SciCom KW - Whole genome sequencing (WGS) AB -

In October 2021 the Scientific Committee of the Federal Agency for the Safety of the Food Chain (FASFC) published the opinion 18-2021 regarding whole genome sequencing (WGS) for the detection of foodborne outbreaks and bacterial risk assessment. This opinion was prepared using a self-tasking mandate and published after public consultation.

The added value of WGS for the detection of foodborne outbreaks and bacterial risk assessment has been demonstrated on many occasions in the last couple of years. Therefore, the implementation of WGS in the Belgian context needs some serious reflection. In the future, WGS will largely replace the various older typing methods in the field of bacterial food safety investigation, due to its high discriminatory power and the general use at the international level. Therefore, the Scientific Committee advises the FASFC to gradually make the transition to WGS for the analysis of food isolates.

While the advantages of the WGS method are considerable, some challenges still need to be taken into account for routine and uniform implementation. As with the implementation of any method the validations of the WGS methodology requires a significant effort. Furthermore, the interpretation of the results should be done with care. Therefore, in outbreak investigations it is recommended that WGS-based results be interpreted by a multidisciplinary team (microbiologists, molecular biologists, bioinformaticians, epidemiologists) with sufficient expertise. Only by putting the WGS-based results, the epidemiological evidence and the strains metadata together can a hypothesis be formulated. Further care should be given to the communication of these interpretations or hypotheses to the different actors (competent authority, food business operator, consumer). Finally, effort should be made to facilitate data sharing in an appropriate way.

JF - 26th Conference on Food Microbiology PB - Belgian Society for Food Microbiology CY - Brussels, Belgium ER - TY - Generic T1 - The key role of WGS-based surveillance of Listeria monocytogenes in both human and food isolates in outbreak investigations Y1 - 2022 A1 - An Van den Bossche A1 - Sigrid C.J. De Keersmaecker A1 - Wesley Mattheus A1 - Nancy Roosens A1 - Bert Bogaerts A1 - Kevin Vanneste A1 - Koenraad Van Hoorde A1 - Bavo Verhaegen KW - cgMLST KW - Listeria monocytogenes KW - NGS KW - outbreak JF - 26th Conference on Food Microbiology PB - BSFM CY - Brussels, Belgium CP - BSFM ER - TY - JOUR T1 - Listeria monocytogenes isolates from Cornu aspersum snails: Whole genome-based characterization and host-pathogen interactions in a snail infection model. JF - Fish Shellfish Immunol Y1 - 2022 A1 - Kotzamanidis, Charalampos A1 - Malousi, Andigoni A1 - Esmeralda Dushku A1 - Alexandre Dobly A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Karathodorou, Argyro A1 - Alexandra Staikou A1 - Antonios Zdragas A1 - Minas Yiangou KW - Animals KW - Host-Pathogen Interactions KW - Listeria monocytogenes KW - Listeriosis KW - Meat KW - whole genome sequencing AB -

Even though Listeria monocytogenes is an extensive-studied foodborne pathogen, genome analysis of isolates from snails that may represent a reservoir of L. monocytogenes are still scarce. Here, we use whole-genome sequencing (WGS) to assess the genomic diversity of hypervirulent, virulent and non-virulent phenotypes of 15 L. monocytogenes isolated from snails to unveil their survival, virulence, and host-pathogen mechanisms of interactions in a snail infection model. Most of isolates (66.7%) were characterized as multidrug resistant (MDR) and belonged to clonal complexes (CCs) which are strongly associated with cases of human infection. All isolates contained intact genes associated with invasion and infection while hypervirulent isolates are adapted to host environment, possessing genes which are involved in teichoic acid biosynthesis, peptidoglycan modification and biofilm formation, correlating with their tolerance to haemolymph plasma phenotype and biofilm formation ability. A snail infection model showed that hypervirulent isolates triggered programmed host cell death pathway by increasing up to 30% the circulating apoptotic hemocytes in combination with induced nitrate production and reactive oxygen species (ROS) generation in snails' haemolymph. In contrast, the administration of the non-virulent strain which possesses a truncated mogR gene that regulates flagellar motility gene expression led only to an increase of necrotic non-apoptotic cells. Overall, this study provides significant insights into the genetic diversity of L. monocytogenes from snails, the genomic features of them linked to their hypervirulent/non-virulent phenotype, and the mechanisms of host-pathogen interactions.

VL - 123 M3 - 10.1016/j.fsi.2022.03.028 ER - TY - JOUR T1 - Metagenomic Characterization of Multiple Genetically Modified Bacillus Contaminations in Commercial Microbial Fermentation Products JF - Life Y1 - 2022 A1 - Jolien D'aes A1 - Marie-Alice Fraiture A1 - Bert Bogaerts A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Kevin Vanneste KW - Bacillus KW - food enzyme KW - genetically modified microorganisms (GMM) KW - hybrid genome assembly KW - metagenomic shotgun sequencing AB -

Genetically modified microorganisms (GMM) are frequently employed for manufacturing microbial fermentation products such as food enzymes or vitamins. Although the fermentation product is required to be pure, GMM contaminations have repeatedly been reported in numerous commercial microbial fermentation produce types, leading to several rapid alerts at the European level. The aim of this study was to investigate the added value of shotgun metagenomic high-throughput sequencing to confirm and extend the results of classical analysis methods for the genomic characterization of unauthorized GMM. By combining short- and long-read metagenomic sequencing, two transgenic constructs were characterized, with insertions of alpha-amylase genes originating from B. amyloliquefaciens and B. licheniformis, respectively, and a transgenic construct with a protease gene insertion originating from B. velezensis, which were all present in all four investigated samples. Additionally, the samples were contaminated with up to three unculturable Bacillus strains, carrying genetic modifications that may hamper their ability to sporulate. Moreover, several samples contained viable Bacillus strains. Altogether these contaminations constitute a considerable load of antimicrobial resistance genes, that may represent a potential public health risk. In conclusion, our study showcases the added value of metagenomics to investigate the quality and safety of complex commercial microbial fermentation products.

VL - 12 CP - 12 M3 - 10.3390/life12121971 ER - TY - JOUR T1 - Metagenomics to Detect and Characterize Viruses in Food Samples at Genome Level? Lessons Learnt from a Norovirus Study JF - Foods Y1 - 2022 A1 - Florence E Buytaers A1 - Bavo Verhaegen A1 - Mathieu Gand A1 - Jolien D'aes A1 - Kevin Vanneste A1 - Nancy Roosens A1 - Kathleen Marchal A1 - Sarah Denayer A1 - Sigrid C.J. De Keersmaecker VL - 11 CP - 21 M3 - 10.3390/foods11213348 ER - TY - JOUR T1 - Noninvasive integrative approach applied to children in the context of recent air pollution exposure demonstrates association between fractional exhaled nitric oxide (FeNO) and urinary CC16. JF - Environ Res Y1 - 2022 A1 - S. Nauwelaerts A1 - Nina Van Goethem A1 - Koen De Cremer A1 - Natalia Bustos Sierra A1 - Jordy Vercauteren A1 - Christophe Stroobants A1 - Bernard, Alfred A1 - Nawrot, Tim A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker AB -

Exposure to the air pollutant particulate matter (PM) is associated with increased risks of respiratory diseases and enhancement of airway inflammation in children. In the context of large scale air pollution studies, it can be challenging to measure fractional exhaled nitric oxide (FeNO) as indicator of lung inflammation. Urinary CC16 (U-CC16) is a potential biomarker of increased lung permeability and toxicity, increasing following short-term PM exposure. The single nucleotide polymorphism (SNP) CC16 G38A (rs3741240) affects CC16 levels and respiratory health. Our study aimed at assessing the use of U-CC16 (incl. CC16 G38A from saliva) as potential alternative for FeNO by investigating their mutual correlation in children exposed to PM. Samples from a small-scale study conducted in 42 children from urban (n = 19) and rural (n = 23) schools examined at two time points, were analysed. When considering recent (lag1) low level exposure to PM as air pollution measurement, we found that U-CC16 was positively associated with FeNO (β = 0.23; 95% CI [-0.01; 0.47]; p = 0.06) in an adjusted analysis using a linear mixed effects model. Further, we observed a positive association between PM and FeNO (β = 0.56; 95% CI [0.02; 1.09]; p = 0.04) and higher FeNO in urban school children as compared to rural school children (β = 0.72; 95% CI [0.12; 1.31]; p = 0.02). Although more investigations are needed, our results suggest that inflammatory responses evidenced by increased FeNO are accompanied by potential increased lung epithelium permeability and injury, evidenced by increased U-CC16. In future large scale studies, where FeNO measurement is less feasible, the integrated analysis of U-CC16 and CC16 G38A, using noninvasive samples, might be a suitable alternative to assess the impact of air pollution exposure on the respiratory health of children, which is critical for policy development at population level.

VL - 216 CP - Pt 1 M3 - 10.1016/j.envres.2022.114441 ER - TY - RPRT T1 - Optimised long-read sequence bioinformatics tool for the analysis of the resistome and microbial communities Y1 - 2022 A1 - Manal AbuOun A1 - Saria Otani A1 - Indre Navickaite A1 - Mathieu Gand A1 - Bram Bloemen A1 - Kevin Vanneste A1 - Valeria Michelacci A1 - Bosco Rodríguez Matamoros A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - Antimicrobial resistance KW - bioinformatics KW - Long read metagenomic sequencing KW - Microbial community KW - Resistome PB - OpenAIRE CY - Zenodo UR - https://doi.org/10.5281/zenodo.7433258 M3 - https://doi.org/10.5281/zenodo.7433258 ER - TY - JOUR T1 - Optimization and Application of a Multiplex Digital PCR Assay for the Detection of SARS-CoV-2 Variants of Concern in Belgian Influent Wastewater. JF - Viruses Y1 - 2022 A1 - Tim Boogaerts A1 - Siel Van den Bogaert A1 - Laura Van Poelvoorde A1 - Diala El Masri A1 - Naomi De Roeck A1 - Nancy Roosens A1 - Marie Lesenfants A1 - Lies Lahousse A1 - Koenraad Van Hoorde A1 - Alexander L N van Nuijs A1 - Peter Delputte KW - Belgium KW - COVID-19 KW - Humans KW - Multiplex Polymerase Chain Reaction KW - Pandemics KW - RNA, Viral KW - SARS-CoV-2 KW - Waste water AB -

Since the beginning of the COVID-19 pandemic, the wastewater-based epidemiology (WBE) of SARS-CoV-2 has been used as a complementary indicator to follow up on the trends in the COVID-19 spread in Belgium and in many other countries. To further develop the use of WBE, a multiplex digital polymerase chain reaction (dPCR) assay was optimized, validated and applied for the measurement of emerging SARS-CoV-2 variants of concern (VOC) in influent wastewater (IWW) samples. Key mutations were targeted in the different VOC strains, including SΔ69/70 deletion, N501Y, SΔ241 and SΔ157. The presented bioanalytical method was able to distinguish between SARS-CoV-2 RNA originating from the wild-type and B.1.1.7, B.1.351 and B.1.617.2 variants. The dPCR assay proved to be sensitive enough to detect low concentrations of SARS-CoV-2 RNA in IWW since the limit of detection of the different targets ranged between 0.3 and 2.9 copies/µL. This developed WBE approach was applied to IWW samples originating from different Belgian locations and was able to monitor spatio-temporal changes in the presence of targeted VOC strains in the investigated communities. The present dPCR assay developments were realized to bring added-value to the current national WBE of COVID-19 by also having the spatio-temporal proportions of the VoC in presence in the wastewaters.

VL - 14 CP - 3 M3 - 10.3390/v14030610 ER - TY - JOUR T1 - Population Analysis of O26 Shiga Toxin-Producing Causing Hemolytic Uremic Syndrome in Italy, 1989-2020, Through Whole Genome Sequencing. JF - Front Cell Infect Microbiol Y1 - 2022 A1 - Valeria Michelacci A1 - Margherita Montalbano Di Filippo A1 - Federica Gigliucci A1 - Silvia Arancia A1 - Paola Chiani A1 - Fabio Minelli A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker A1 - Bert Bogaerts A1 - Kevin Vanneste A1 - Stefano Morabito KW - Escherichia coli Infections KW - Escherichia coli Proteins KW - Hemolytic-Uremic Syndrome KW - Humans KW - ITALY KW - Multilocus Sequence Typing KW - Shiga-Toxigenic Escherichia coli KW - whole genome sequencing AB -

Shiga toxin-producing (STEC) belonging to the O26 serogroup represent an important cause of Hemolitic Uremic Syndrome (HUS) in children worldwide. The localization of STEC virulence genes on mobile genetic elements allowed the emergence of clones showing different assets of this accessory genomic fraction. A novel O26 STEC clone belonging to Sequence Type (ST) 29 and harboring , and plasmid-borne genes has emerged and spread in Europe since the mid-1990s, while another ST29 clone positive for and lacking plasmid-borne virulence genes was recently described as emerging in France. In Italy, O26 has been the most frequently detected STEC serogroup from HUS cases since the late 1990s. In this study we describe the genomic characterization and population structure of 144 O26 STEC strains isolated from human sources in Italy in the period 1989-2020. A total of 89 strains belonged to ST21, 52 to ST29, two to ST396 and one to ST4944. ST29 strains started to be isolated from 1999. 24 strains were shown to harbour , alone (n=20) or in combination with (n=4). The majority of the strains (n=118) harbored genes only and the two ST396 strains harbored . A Hierarchical Clustering on Principal Components (HCPC) analysis, based on the detection of accessory virulence genes, antimicrobial resistance (AMR) genes and plasmid replicons, classified the strains in seven clusters identified with numbers from 1 to 7, containing two, 13, 39, 63, 16, 10 and one strain, respectively. The majority of the genetic features defining the clusters corresponded to plasmid-borne virulence genes, AMR genes and plasmid replicons, highlighting specific assets of plasmid-borne features associated with different clusters. Core genome Multi Locus Sequence Typing grouped ST21 and ST29 strains in three clades each, with each ST29 clade exactly corresponding to one HCPC cluster. Our results showed high conservation of either the core or the accessory genomic fraction in populations of ST29 O26 STEC, differently from what observed in ST21 strains, suggesting that a different selective pressure could drive the evolution of different populations of these pathogens possibly involving different ecological niches.

VL - 12 M3 - 10.3389/fcimb.2022.842508 ER - TY - RPRT T1 - Report on an initial assessment of long-read sequencing for metagenome (community and AMR) analysis (defined community, spiked and real simple and complex matrices) and comparison to current short-read standard Y1 - 2022 A1 - Sigrid C.J. De Keersmaecker A1 - Mathieu Gand A1 - Indre Navickaite A1 - Josephine Gruetzke A1 - Lee-Julia Bartsch A1 - Saria Otani A1 - Giuliano Garofolo A1 - Marco Di Domenico A1 - Valeria Michelacci A1 - Bosco R Matamoros A1 - Carlus Deneke A1 - Lisa Di Marcantonio A1 - Francesca Marotta A1 - Jennie Fischer A1 - Bram Bloemen A1 - Kevin Vanneste A1 - Nancy Roosens A1 - Michael Brouwer A1 - Quillan Dijkstra A1 - Søren Overballe-Petersen A1 - Søren Persson A1 - Astrid Rasmussen A1 - Bruno Gonzalez-Zorn A1 - Manal AbuOun KW - Antimicrobial resistance KW - Bacterial community KW - Long read metagenomics KW - Metagenome PB - OpenAIRE CY - Zenodo UR - https://doi.org/10.5281/zenodo.7429361 M3 - https://doi.org/10.5281/zenodo.7429361 ER - TY - RPRT T1 - Report on initial findings of on-site diagnostic tests and IT solutions for human clinical, veterinary and environmental samples for early warning of emerging resistant pathogens Y1 - 2022 A1 - Manal AbuOun A1 - Indre Navickaite A1 - Thomas I. Wilkes A1 - Marco Di Domenico A1 - Giuliano Garofolo A1 - Bram Bloemen A1 - Mathieu Gand A1 - Nancy Roosens A1 - Kevin Vanneste A1 - Sigrid C.J. De Keersmaecker A1 - Quillan Dijkstra A1 - Saria Otani A1 - Michael Brouwer KW - Long read metagenomics KW - Metagenome KW - On-site metagenomics PB - OpenAIRE CY - Zenodo UR - https://doi.org/10.5281/zenodo.7433281 M3 - https://doi.org/10.5281/zenodo.7433281 ER - TY - RPRT T1 - Report on the feasibility of using Hi-C sequencing for metagenomics to define the context of AMR genes Y1 - 2022 A1 - Sigrid C.J. De Keersmaecker A1 - Josephine Grützke A1 - Carlus Deneke A1 - Jennie Fischer A1 - Lee Julia Bartsch A1 - Bram Bloemen A1 - Kevin Vanneste A1 - Jolien D'aes A1 - Nancy Roosens A1 - Manal AbuOun KW - Antimicrobial resistance KW - Hi-C metagenomics PB - OpenAIRE CY - Zenodo UR - https://zenodo.org/record/7433351#.Y7aj4HbMKM8 M3 - 10.5281/zenodo.7433350 ER - TY - Generic T1 - A shotgun metagenomics approach to detect and characterize unauthorized genetically modified microorganisms in microbial fermentation products Y1 - 2022 A1 - Florence E Buytaers A1 - Marie-Alice Fraiture A1 - Bas Berbers A1 - Els Vandermassen A1 - Stefan Hoffman A1 - N. Papazova A1 - Kevin Vanneste A1 - Kathleen Marchal A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker JF - ONE Conference 2022 CP - EFSA ER - TY - Generic T1 - Shotgun metagenomics as a ONE Health tool for better protecting human health Y1 - 2022 A1 - Mathieu Gand A1 - Florence E Buytaers A1 - Bram Bloemen A1 - Assia Saltykova A1 - Sarah Denayer A1 - Bavo Verhaegen A1 - Wesley Mattheus A1 - Denis Piérard A1 - Bert Bogaerts A1 - Kathleen Marchal A1 - The FARMED consortium A1 - Nancy Roosens A1 - Kevin Vanneste A1 - Sigrid C.J. De Keersmaecker AB -

Introduction: Open approaches to identify all biological material in a sample without a priori knowing what to look for, could transform the way specific key public health questions can be addressed, in a ONE Health context. Metagenomics is defined as the study of all genetic material within a sample, using sequencing technologies. Shotgun metagenomics is capable of detecting a wide range of microorganisms in a sample, as well as their composing genes such as virulence or antimicrobial resistance (AMR) genes, without prior knowledge, nor the need to isolate. Such approaches could offer efficient and fast solutions to limitations faced by conventional methods in e.g. the field of pathogen detection/characterization along the food chain, starting from the food production environment (including detection of AMR genes) until foodborne outbreak investigation and clinical/veterinary diagnostics. This could also allow a better understanding of infectious diseases and accurate risk (safety) assessment in food safety issues. However, before it can be used in these settings, this approach should be appropriately developed and validated from sampling and DNA extraction to sequencing and data analysis.

Methodology: Third generation sequencing technologies have had a breakthrough with the launch of nanopore sequencing, which through the production of long sequencing reads, portability and real-time data generation, represents a disruptive innovation for microbiology. Compared to short read sequencing (Illumina), this technology allows to unambiguously detect and scaffold microbial genes to their host chromosomes, even for complex metagenomics samples, allowing taxonomic classification up to an unprecedented sensitivity, including the identification of linked specific genes, such as AMR or virulence genes. We are developing and optimizing the protocols (from DNA extraction to data analysis) for sensitive metagenomics, by analyzing various materials collected in food-producing environments, and spiked with defined mock communities, composed of different bacterial species in various concentrations and carrying AMR and/or virulence genes, taking the specific need of each application into account. Moreover, the short read and long read approaches are being compared to each other, and also to the results obtained with conventional methods.

Results: A commercial kit was selected and optimized for the extraction of sufficient high-molecular weight DNA, to match the needs for long-read sequencing. The analysis of animal and environmental samples collected at a production farm, subsequently spiked with bacterial species present at various concentrations and carrying AMR genes, allowed to identify the spiked species and to make the link with AMR genes detected in the same sample when found on the same reads used for taxonomic identification, except for species present at low concentrations. Comparable results were obtained using short-read sequencing, without being able to link the identified species with the detected AMR genes. Our approach was also successful on artificially contaminated food samples. It allowed the same characterization of the foodborne pathogen (serotyping, virulence genes, relatedness to other cases) as the conventional methods used in reference laboratories, however without isolation and in a shorter time-frame. This could be achieved to the strain level even in samples with several E. coli strains. The method was also used to solve a Salmonella food-borne outbreak that occurred in Belgium in 2019.

Discussion: By applying the shotgun metagenomics approach to specific case studies, we delivered proof-of-concepts and demonstrated its added value compared to the conventional methods. We proved that it can characterize a bacterial pathogen to the strain level in spiked food as well as in the context of a real outbreak, and resolve the investigation in a faster time frame. EFSA called for such proof-of-concepts in a recent opinion about the use of metagenomics for food safety.  Additionally, we showed that the use of long-read sequencing can help to achieve a higher level of resolution by identifying specific bacteria and the AMR genes present in their genomes, with only one analysis. Future studies will explore at the technical level how this new technology can be transferred for fast, easy and direct use on-site, in a food-producing environment. This will open up ample opportunities for future research in the different fields, and even beyond, where the metagenomics approach would then be a well-established tool to be used to address specific public health questions at a more detailed and extended/comprehensive level, contributing to better protection of human health.

JF - EFSA One CP - EFSA ER - TY - JOUR T1 - Urinary CC16, a potential indicator of lung integrity and inflammation, increases in children after short-term exposure to PM/PM and is driven by the CC16 38GG genotype. JF - Environ Res Y1 - 2022 A1 - S. Nauwelaerts A1 - Nina Van Goethem A1 - Berta Tenas Ureña A1 - Koen De Cremer A1 - Bernard, Alfred A1 - Nelly D Saenen A1 - Nawrot, Tim S A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - Air Pollutants KW - Biomarkers KW - Child KW - environmental exposure KW - Genotype KW - Humans KW - Inflammation KW - Lung KW - Particulate Matter KW - Retinol-Binding Proteins, Plasma KW - Uteroglobin AB -

Particular matter (PM) exposure is a big hazard for public health, especially for children. Serum CC16 is a well-known biomarker of respiratory health. Urinary CC16 (U-CC16) can be a noninvasive alternative, albeit requiring adequate adjustment for renal handling. Moreover, the SNP CC16 G38A influences CC16 levels. This study aimed to monitor the effect of short-term PM exposure on CC16 levels, measured noninvasively in schoolchildren, using an integrative approach. We used a selection of urine and buccal DNA samples from 86 children stored in an existing biobank. Using a multiple reaction monitoring method, we measured U-CC16, as well as RBP4 (retinol binding protein 4) and β2M (beta-2-microglobulin), required for adjustment. Buccal DNA samples were used for CC16 G38A genotyping. Linear mixed-effects models were used to find relevant associations between U-CC16 and previously obtained data from recent daily PM ≤ 2.5 or 10 μm exposure (PM, PM) modeled at the child's residence. Our study showed that exposure to low PM at the child's residence (median levels 18.9 μg/m³ (PM) and 23.6 μg/m³ (PM)) one day before sampling had an effect on the covariates-adjusted U-CC16 levels. This effect was dependent on the CC16 G38A genotype, due to its strong interaction with the association between PM levels and covariates-adjusted U-CC16 (P = 0.024 (PM); P = 0.061 (PM)). Only children carrying the 38GG genotype showed an increase of covariates-adjusted U-CC16, measured 24h after exposure, with increasing PM and PM (β = 0.332; 95% CI: 0.110 to 0.554 and β = 0.372; 95% CI: 0.101 to 0.643, respectively). To the best of our knowledge, this is the first study using an integrative approach to investigate short-term PM exposure of children, using urine to detect early signs of pulmonary damage, and taking into account important determinants such as the genetic background and adequate adjustment of the measured biomarker in urine.

VL - 212 CP - Pt B M3 - 10.1016/j.envres.2022.113272 ER - TY - JOUR T1 - Whole-Genome Sequence Approach and Phylogenomic Stratification Improve the Association Analysis of Mutations With Patient Data in Influenza Surveillance JF - Frontiers in Microbiology Y1 - 2022 A1 - Laura Van Poelvoorde A1 - Kevin Vanneste A1 - Sigrid C.J. De Keersmaecker A1 - Isabelle Thomas A1 - Nina Van Goethem A1 - Steven Van Gucht A1 - Xavier Saelens A1 - Nancy Roosens VL - 13 M3 - 10.3389/fmicb.2022.809887 ER - TY - JOUR T1 - Whole-Genome Sequence Approach and Phylogenomic Stratification Improve the Association Analysis of Mutations With Patient Data in Influenza Surveillance. JF - Front Microbiol Y1 - 2022 A1 - Laura Van Poelvoorde A1 - Kevin Vanneste A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens ED - Isabelle Thomas ED - Steven Van Gucht AB -

Each year, seasonal influenza results in high mortality and morbidity. The current classification of circulating influenza viruses is mainly focused on the hemagglutinin gene. Whole-genome sequencing (WGS) enables tracking mutations across all influenza segments allowing a better understanding of the epidemiological effects of intra- and inter-seasonal evolutionary dynamics, and exploring potential associations between mutations across the viral genome and patient's clinical data. In this study, mutations were identified in 253 Influenza A (H3N2) clinical isolates from the 2016-2017 influenza season in Belgium. As a proof of concept, available patient data were integrated with this genomic data, resulting in statistically significant associations that could be relevant to improve the vaccine and clinical management of infected patients. Several mutations were significantly associated with the sampling period. A new approach was proposed for exploring mutational effects in highly diverse Influenza A (H3N2) strains through considering the viral genetic background by using phylogenetic classification to stratify the samples. This resulted in several mutations that were significantly associated with patients suffering from renal insufficiency. This study demonstrates the usefulness of using WGS data for tracking mutations across the complete genome and linking these to patient data, and illustrates the importance of accounting for the viral genetic background in association studies. A limitation of this association study, especially when analyzing stratified groups, relates to the number of samples, especially in the context of national surveillance of small countries. Therefore, we investigated if international databases like GISAID may help to verify whether observed associations in the Belgium A (H3N2) samples, could be extrapolated to a global level. This work highlights the need to construct international databases with both information of viral genome sequences and patient data.

VL - 13 M3 - 10.3389/fmicb.2022.809887 ER - TY - JOUR T1 - Application of a strain-level shotgun metagenomics approach on food samples: resolution of the source of a Salmonella food-borne outbreak JF - Microbial Genomics Y1 - 2021 A1 - Florence E Buytaers A1 - Assia Saltykova A1 - Wesley Mattheus A1 - Bavo Verhaegen A1 - Nancy Roosens A1 - Kevin Vanneste A1 - Valeska Laisnez A1 - Hammami, Naïma A1 - Pochet, Brigitte A1 - Vera Cantaert A1 - Marchal, Kathleen A1 - Sarah Denayer A1 - Sigrid C.J. De Keersmaecker KW - food surveillance KW - Metagenomics KW - outbreak KW - Salmonella KW - SNP analysis KW - strain-level AB -

Food-borne outbreak investigation currently relies on the time-consuming and challenging bacterial isolation from food, to be able to link food-derived strains to more easily obtained isolates from infected people. When no food isolate can be obtained, the source of the outbreak cannot be unambiguously determined. Shotgun metagenomics approaches applied to the food samples could circumvent this need for isolation from the suspected source, but require downstream strain-level data analysis to be able to accurately link to the human isolate. Until now, this approach has not yet been applied outside research settings to analyse real food-borne outbreak samples. In September 2019, a Salmonella outbreak occurred in a hotel school in Bruges, Belgium, affecting over 200 students and teachers. Following standard procedures, the Belgian National Reference Center for human salmonellosis and the National Reference Laboratory for Salmonella in food and feed used conventional analysis based on isolation, serotyping and MLVA (multilocus variable number tandem repeat analysis) comparison, followed by whole-genome sequencing, to confirm the source of the contamination over 2 weeks after receipt of the sample, which was freshly prepared tartar sauce in a meal cooked at the school. Our team used this outbreak as a case study to deliver a proof of concept for a short-read strain-level shotgun metagenomics approach for source tracking. We received two suspect food samples: the full meal and some freshly made tartar sauce served with this meal, requiring the use of raw eggs. After analysis, we could prove, without isolation, that Salmonella was present in both samples, and we obtained an inferred genome of a Salmonella enterica subsp. enterica serovar Enteritidis that could be linked back to the human isolates of the outbreak in a phylogenetic tree. These metagenomics-derived outbreak strains were separated from sporadic cases as well as from another outbreak circulating in Europe at the same time period. This is, to our knowledge, the first Salmonella food-borne outbreak investigation uniquely linking the food source using a metagenomics approach and this in a fast time frame.

VL - 7 CP - 4 M3 - 10.1099/mgen.0.000547 ER - TY - JOUR T1 - A Bioinformatics Whole-Genome Sequencing Workflow for Clinical Mycobacterium tuberculosis Complex Isolate Analysis, Validated Using a Reference Collection Extensively Characterized with Conventional Methods and In Silico Approaches JF - J Clin Microbiol Y1 - 2021 A1 - Bert Bogaerts A1 - Thomas Delcourt A1 - Karine Soetaert A1 - Samira Boarbi A1 - Pieter-Jan Ceyssens A1 - Raf Winand A1 - Julien Van Braekel A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Marchal, Kathleen A1 - Vanessa Mathys A1 - Kevin Vanneste KW - Antimicrobial resistance KW - Mycobacterium tuberculosis KW - national reference center KW - public health KW - single nucleotide polymorphism KW - VALIDATION KW - whole genome sequencing AB -

The use of whole genome sequencing (WGS) for routine typing of bacterial isolates has increased substantially in recent years. For (MTB), in particular, WGS has the benefit of drastically reducing the time to generate results compared to most conventional phenotypic methods. Consequently, a multitude of solutions for analyzing WGS MTB data have been developed, but their successful integration in clinical and national reference laboratories is hindered by the requirement for their validation, for which a consensus framework is still largely absent. We developed a bioinformatics workflow for (Illumina) WGS-based routine typing of MTB Complex (MTBC) member isolates allowing complete characterization including (sub)species confirmation and identification (16S, /RD, , Single Nucleotide Polymorphism (SNP)-based antimicrobial resistance (AMR) prediction, and pathogen typing (spoligotyping, SNP barcoding, and core genome MultiLocus Sequence Typing). Workflow performance was validated on a per-assay basis using a collection of 238 in-house sequenced MTBC isolates, extensively characterized with conventional molecular biology-based approaches supplemented with public data. For SNP-based AMR prediction, results from molecular genotyping methods were supplemented with modified datasets allowing to greatly increase the set of evaluated mutations. The workflow demonstrated very high performance with performance metrics >99% for all assays, except for spoligotyping where sensitivity dropped to ∼90%. The validation framework for our WGS-based bioinformatics workflow can aid standardization of bioinformatics tools by the MTB community and other SNP-based applications regardless of the targeted pathogen(s). The bioinformatics workflow is available for academic and non-profit usage through the Galaxy instance of our institute at https://galaxy.sciensano.be.

M3 - 10.1128/JCM.00202-21 ER - TY - JOUR T1 - Case Report: Multidrug Resistant Raoultella ornithinolytica in a Septicemic Calf JF - Front Vet Sci Y1 - 2021 A1 - Mathilde L Pas A1 - Kevin Vanneste A1 - Jade Bokma A1 - Van Driessche, Laura A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Haesebrouck, Freddy A1 - Boyen, Filip A1 - Pardon, Bart AB -

Sepsis is a frequent life-threatening condition in young calves, requiring rapid broad spectrum and bactericidal therapy to maximize survival chances. Few studies have identified and characterized bacteria involved in sepsis in calves. This report demonstrates the involvement of a multidrug resistant , an emerging pathogen in human medicine, in a calf with suspected sepsis. was identified by MALDI-TOF MS from blood cultures of a critically ill calf. Susceptibility testing showed phenotypic resistance against ampicillin, gentamicin, potentiated sulphonamides, streptomycin, tetracyclines and intermediate susceptibility for enrofloxacin. Whole genome sequencing confirmed identification as and the multidrug resistant character of the isolate. Antimicrobial resistance genes acting against aminoglycosides, beta-lactam antibiotics, fosfomycin, quinolones, sulphonamides, trimethoprim and tetracyclines were found. The calf recovered after empirical parenteral therapy with enrofloxacin and sodium penicillin for seven days. Ancillary therapy consisted of fluid therapy, ketoprofen and doxapram hydrochloride. To the authors' knowledge, this is the first report characterizing a multidrug resistant isolate from blood culture in cattle. It is currently unknown whether animals and farms may act as reservoirs for multidrug resistant strains.

VL - 8 M3 - 10.3389/fvets.2021.631716 ER - TY - JOUR T1 - Characterization of Genetically Modified Microorganisms Using Short- and Long-Read Whole-Genome Sequencing Reveals Contaminations of Related Origin in Multiple Commercial Food Enzyme Products. JF - Foods Y1 - 2021 A1 - Jolien D'aes A1 - Marie-Alice Fraiture A1 - Bert Bogaerts A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Kevin Vanneste AB -

Despite their presence being unauthorized on the European market, contaminations with genetically modified (GM) microorganisms have repeatedly been reported in diverse commercial microbial fermentation produce types. Several of these contaminations are related to a GM used to synthesize a food enzyme protease, for which genomic characterization remains currently incomplete, and it is unknown whether these contaminations have a common origin. In this study, GM isolates from multiple food enzyme products were characterized by short- and long-read whole-genome sequencing (WGS), demonstrating that they harbor a free recombinant pUB110-derived plasmid carrying antimicrobial resistance genes. Additionally, single-nucleotide polymorphism (SNP) and whole-genome based comparative analyses showed that the isolates likely originate from the same parental GM strain. This study highlights the added value of a hybrid WGS approach for accurate genomic characterization of GMM (e.g., genomic location of the transgenic construct), and of SNP-based phylogenomic analysis for source-tracking of GMM.

VL - 10 CP - 11 M3 - 10.3390/foods10112637 ER - TY - JOUR T1 - Coverage of the national surveillance system for human Salmonella infections, Belgium, 2016-2020. JF - PLoS One Y1 - 2021 A1 - Nina Van Goethem A1 - An Van den Bossche A1 - Pieter-Jan Ceyssens A1 - Adrien Lajot A1 - Wim Coucke A1 - Kris Vernelen A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker A1 - Dieter Van Cauteren A1 - Wesley Mattheus KW - Belgium KW - Diagnostic Tests, Routine KW - Disease Outbreaks KW - Humans KW - Public Health Surveillance KW - Salmonella KW - Salmonella Food Poisoning KW - Salmonella Infections KW - whole genome sequencing AB -

INTRODUCTION: The surveillance of human salmonellosis in Belgium is dependent on the referral of human Salmonella isolates to the National Reference Center (NRC). Knowledge of current diagnostic practices and the coverage of the national Salmonella surveillance system are important to correctly interpret surveillance data and trends over time, to estimate the true burden of salmonellosis in Belgium, and to evaluate the appropriateness of implementing whole-genome sequencing (WGS) at this central level.

METHODS: The coverage of the NRC was defined as the proportion of all diagnosed human Salmonella cases in Belgium reported to the NRC and was assessed for 2019 via a survey among all licensed Belgian medical laboratories in 2019, and for 2016-2020 via a capture-recapture study using the Sentinel Network of Laboratories (SNL) as the external source. In addition, the survey was used to assess the impact of the implementation of culture-independent diagnostic tests (CIDTs) at the level of peripheral laboratory sites, as a potential threat to national public health surveillance programs.

RESULTS: The coverage of the NRC surveillance system was estimated to be 83% and 85%, based on the results of the survey and on the two-source capture-recapture study, respectively. Further, the results of the survey indicated a limited use of CIDTs by peripheral laboratories in 2019.

CONCLUSION: Given the high coverage and the limited impact of CIDTs on the referral of isolates, we may conclude that the NRC can confidently monitor the epidemiological situation and identify outbreaks throughout the country. These findings may guide the decision to implement WGS at the level of the NRC and may improve estimates of the true burden of salmonellosis in Belgium.

VL - 16 CP - 8 M3 - 10.1371/journal.pone.0256820 ER - TY - JOUR T1 - Deepening of In Silico Evaluation of SARS-CoV-2 Detection RT-qPCR Assays in the Context of New Variants JF - Genes Y1 - 2021 A1 - Mathieu Gand A1 - Kevin Vanneste A1 - Isabelle Thomas A1 - Steven Van Gucht A1 - Arnaud Capron A1 - Philippe Herman A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - SARS-CoV-2; COVID-19; variants; detection; RT-qPCR; in silico specificity evaluation; bioinformatics tool; WGS data; mismatches; primers and probes AB -

For 1 year now, the world is undergoing a coronavirus disease-2019 (COVID-19) pandemic due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The most widely used method for COVID-19 diagnosis is the detection of viral RNA by RT-qPCR with a specific set of primers and probe. It is important to frequently evaluate the performance of these tests and this can be done first by an in silico approach. Previously, we reported some mismatches between the oligonucleotides of publicly available RT-qPCR assays and SARS-CoV-2 genomes collected from GISAID and NCBI, potentially impacting proper detection of the virus. In the present study, 11 primers and probe sets investigated during the first study were evaluated again with 84,305 new SARS-CoV-2 unique genomes collected between June 2020 and January 2021. The lower inclusivity of the China CDC assay targeting the gene N has continued to decrease with new mismatches detected, whereas the other evaluated assays kept their inclusivity above 99%. Additionally, some mutations specific to new SARS-CoV-2 variants of concern were found to be located in oligonucleotide annealing sites. This might impact the strategy to be considered for future SARS-CoV-2 testing. Given the potential threat of the new variants, it is crucial to assess if they can still be correctly targeted by the primers and probes of the RT-qPCR assays. Our study highlights that considering the evolution of the virus and the emergence of new variants, an in silico (re-)evaluation should be performed on a regular basis. Ideally, this should be done for all the RT-qPCR assays employed for SARS-CoV-2 detection, including also commercial tests, although the primer and probe sequences used in these kits are rarely disclosed, which impedes independent performance evaluation.

VL - 12 CP - 4 M3 - 10.3390/genes12040565 ER - TY - JOUR T1 - Development of a multiplex mass spectrometry method for simultaneous quantification of urinary proteins related to respiratory health JF - Scientific Reports Y1 - 2021 A1 - S. Nauwelaerts A1 - Nancy Roosens A1 - Bernard, Alfred A1 - Sigrid C.J. De Keersmaecker A1 - Koen De Cremer VL - 11 CP - 1 M3 - 10.1038/s41598-021-89068-9 ER - TY - JOUR T1 - Development of a real-time PCR marker targeting a new unauthorized genetically modified microorganism producing protease identified by DNA walking JF - International Journal of Food Microbiology Y1 - 2021 A1 - Marie-Alice Fraiture A1 - Andrea Gobbo A1 - Ugo Marchesi A1 - Daniela Verginelli A1 - N. Papazova A1 - Nancy Roosens KW - Antimicrobial resistance genes KW - DNA walking anchored on pUB110 KW - food and feed safety KW - Genetically modified bacteria KW - Protease KW - real-time PCR detection AB -

A PCR-based DNA walking analysis was performed on a protease product suspected to contain a new unauthorized genetically modified microorganism (GMM). Though the characterization of unnatural associations of sequences between the pUB110 shuttle vector and a Bacillus amyloliquefaciens gene coding for a protease, the presence of the GMM was shown. Based on these sequences of interest, a real-time PCR marker was developed to target specifically the newly discovered GMM, namely GMM protease2. The performance of the real-time PCR marker was assessed in terms of specificity and sensitivity. The applicability of the real-time PCR GMM protease2 marker was also demonstrated on microbial fermentation products. To confirm its use by other GMO enforcement laboratories, the transferability of the in-house validated real-time PCR marker was demonstrated by assays performed by an external laboratory.

VL - 354 M3 - https://doi.org/10.1016/j.ijfoodmicro.2021.109330 ER - TY - JOUR T1 - Development of a real-time PCR marker targeting a new unauthorized genetically modified microorganism producing protease identified by DNA walking. JF - International Journal of Food Microbiology Y1 - 2021 A1 - Marie-Alice Fraiture A1 - U Marchesi A1 - D Verginelli A1 - N. Papazova A1 - Nancy Roosens VL - 354 M3 - https://doi.org/10.1016/j.ijfoodmicro.2021.109330 ER - TY - JOUR T1 - Evaluation of the added value of viral genomic information for predicting severity of influenza infection JF - BMC Infectious Diseases Y1 - 2021 A1 - Nina Van Goethem A1 - Annie Robert A1 - Nathalie Bossuyt A1 - Laura Van Poelvoorde A1 - Sophie Quoilin A1 - Sigrid C.J. De Keersmaecker A1 - Brecht Devleesschauwer A1 - Isabelle Thomas A1 - Kevin Vanneste A1 - Nancy Roosens A1 - Herman Van Oyen VL - 21 CP - 1 M3 - 10.1186/s12879-021-06510-z ER - TY - JOUR T1 - Evaluation of WGS performance for bacterial pathogen characterization with the Illumina technology optimized for time-critical situations. JF - Microb Genom Y1 - 2021 A1 - Bert Bogaerts A1 - Raf Winand A1 - Julien Van Braekel A1 - Stefan Hoffman A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker A1 - Marchal, Kathleen A1 - Kevin Vanneste VL - 7 CP - 11 M3 - 10.1099/mgen.0.000699 ER - TY - JOUR T1 - First detection of a plasmid-encoded New-Delhi metallo-beta-lactamase-1 (NDM-1) producing Acinetobacter baumannii using whole genome sequencing, isolated in a clinical setting in Benin. JF - Ann Clin Microbiol Antimicrob Y1 - 2021 A1 - Carine Yehouenou A1 - Bert Bogaerts A1 - Kevin Vanneste A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker A1 - Marchal, Kathleen A1 - Affolabi, Dissou A1 - Reza Soleimani A1 - Rodriguez-Villalobos, Hector A1 - Van Bambeke, Françoise A1 - Olivia Dalleur A1 - Simon, Anne KW - Acinetobacter baumannii KW - Adult KW - beta-Lactamases KW - Female KW - Humans KW - Plasmids KW - whole genome sequencing AB -

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii is considered a top priority pathogen by the World Health Organization for combatting increasing antibiotic resistance and development of new drugs. Since it was originally reported in Klebsiella pneumoniae in 2009, the quick spread of the bla gene encoding a New-Delhi metallo-beta-lactamase-1 (NDM-1) is increasingly recognized as a serious threat. This gene is usually carried by large plasmids and has already been documented in diverse bacterial species, including A. baumannii. Here, we report the first detection of a NDM-1-producing A. baumannii strain isolated in Benin.

CASE PRESENTATION: A 31-year-old woman was admitted to a surgical unit with a diagnosis of post-cesarean hematoma. An extensively-drug resistant A. baumannii strain solely susceptible to amikacin, colistin and ciprofloxacin, and resistant to several other antibiotics including ceftazidime, imipenem, meropenem, gentamicin, tobramycin, ceftazidime/avibactam, and sulfamethoxazole-trimethoprim, was isolated from the wound. Production of NDM-1 was demonstrated by immunochromatographic testing. Whole genome sequencing of the isolate confirmed the presence of bla, but also antibiotic resistance genes against multiple beta-lactamases and other classes of antibiotics, in addition to several virulence genes. Moreover, the bla gene was found to be present in a Tn125 transposon integrated on a plasmid.

CONCLUSIONS: The discovery of this extensively-drug resistant A. baumannii strain carrying bla in Benin is worrying, especially because of its high potential risk of horizontal gene transfer due to being integrated into a transposon located on a plasmid. Strict control and prevention measures should be taken, once NDM-1 positive A. baumannii has been identified to prevent transfer of this resistance gene to other Enterobacterales. Capacity building is required by governmental agencies to provide suitable antibiotic treatment options and strategies, in combination with strengthening laboratory services for detection and surveillance of this pathogen.

VL - 20 CP - 1 M3 - 10.1186/s12941-020-00411-w ER - TY - JOUR T1 - Food Enzyme Database (FEDA): a web application gathering information about food enzyme preparations available on the European market. JF - Database (Oxford) Y1 - 2021 A1 - Marie Deckers A1 - Julien Van Braekel A1 - Kevin Vanneste A1 - Deforce, Dieter A1 - Marie-Alice Fraiture A1 - Nancy Roosens KW - Databases, Factual KW - food KW - Research Report AB -

Following the European Commission No. 1332/2008 regulation and the consequent necessity of a scientific evaluation of food enzymes (FEs) for their approval for sale on the European Union market, many FE dossiers have been submitted to the European Commission and various documents currently co-exist. In order to centralize all relevant information in one structured location that is easily accessible to support enforcement laboratories and the competent authorities, we developed a web application, called Food Enzyme Database (FEDA). FEDA allows searching and collection of information originating from many different sources in one centralized portal. Queries can be performed using key information types, which include information on the producing company, production source (strain type, genetically modified microorganism status), type of enzyme protein and evaluation status with employed evaluation criteria. The database contains all current publicly available information. Centralizing all information coupled with intuitive searching functionality also allows the generation of general statistics regarding the current market situation. FEDA is open access and is freely available at the following location: https://feda.sciensano.be. Database URL : https://feda.sciensano.be.

VL - 2021 M3 - 10.1093/database/baab060 ER - TY - JOUR T1 - Large diversity of linezolid-resistant isolates discovered in food-producing animals through linezolid selective monitoring in Belgium in 2019. JF - J Antimicrob Chemother Y1 - 2021 A1 - Michaël Timmermans A1 - Bert Bogaerts A1 - Kevin Vanneste A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Carole Kowalewicz A1 - Guillaume Simon A1 - Argudín, M Angeles A1 - Deplano, Ariane A1 - Hallin, Marie A1 - P Wattiau A1 - David Fretin A1 - Denis, Olivier A1 - Cécile Boland AB -

BACKGROUND: Linezolid is a critically important antibiotic used to treat human infections caused by MRSA and VRE. While linezolid is not licensed for food-producing animals, linezolid-resistant (LR) isolates have been reported in European countries, including Belgium.

OBJECTIVES: To: (i) assess LR occurrence in staphylococci and enterococci isolated from different Belgian food-producing animals in 2019 through selective monitoring; and (ii) investigate the genomes and relatedness of these isolates.

METHODS: Faecal samples (n = 1325) and nasal swab samples (n = 148) were analysed with a protocol designed to select LR bacteria, including a 44-48 h incubation period. The presence of LR chromosomal mutations, transferable LR genes and their genetic organizations and other resistance genes, as well as LR isolate relatedness (from this study and the NCBI database) were assessed through WGS.

RESULTS: The LR rate differed widely between animal host species, with the highest rates occurring in nasal samples from pigs and sows (25.7% and 20.5%, respectively) and faecal samples from veal calves (16.4%). WGS results showed that LR determinants are present in a large diversity of isolates circulating in the agricultural sector, with some isolates closely related to human isolates, posing a human health risk.

CONCLUSIONS: LR dedicated monitoring with WGS analysis could help to better understand the spread of LR. Cross-selection of LR transferable genes through other antibiotic use should be considered in future action plans aimed at combatting antimicrobial resistance and in future objectives for the rational use of antibiotics in a One Health perspective.

M3 - 10.1093/jac/dkab376 ER - TY - JOUR T1 - Large diversity of linezolid-resistant isolates discovered in food-producing animals through linezolid selective monitoring in Belgium in 2019 JF - Journal of Antimicrobial Chemotherapy Y1 - 2021 A1 - Michaël Timmermans A1 - Bert Bogaerts A1 - Kevin Vanneste A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Carole Kowalewicz A1 - Guillaume Simon A1 - Maria A Argudín A1 - Deplano, Ariane A1 - Hallin, Marie A1 - P Wattiau A1 - David Fretin A1 - Denis, Olivier A1 - Cécile Boland VL - 77 CP - 1 M3 - 10.1093/jac/dkab376 ER - TY - JOUR T1 - Phylogenomic Investigation of Increasing Fluoroquinolone Resistance among Belgian Cases of Shigellosis between 2013 and 2018 Indicates Both Travel-Related Imports and Domestic Circulation. JF - Microorganisms Y1 - 2021 A1 - Bert Bogaerts A1 - Raf Winand A1 - Julien Van Braekel A1 - Wesley Mattheus A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Marchal, Kathleen A1 - Kevin Vanneste A1 - Pieter-Jan Ceyssens AB -

Shigellosis is an acute enteric infection caused mainly by the species and . Since surveillance of these pathogens indicated an increase in ciprofloxacin-resistant samples collected in Belgium between 2013 and 2018, a subset of 148 samples was analyzed with whole genome sequencing (WGS) to investigate their dispersion and underlying genomic features associated with ciprofloxacin resistance. A comparison between observed phenotypes and WGS-based resistance prediction to ciprofloxacin revealed perfect correspondence for all samples. Core genome multi-locus sequence typing and single nucleotide polymorphism-typing were used for phylogenomic investigation to characterize the spread of these infections within Belgium, supplemented with data from international reference collections to place the Belgian isolates within their global context. For , substantial diversity was observed with ciprofloxacin-resistant isolates assigned to several phylogenetic groups. Besides travel-related imports, several clusters of highly similar Belgian isolates could not be linked directly to international travel suggesting the presence of domestically circulating strains. For , Belgian isolates were all limited to lineage III, and could often be traced back to travel to countries in Asia and Africa, sometimes followed by domestic circulation. For both species, several clusters of isolates obtained exclusively from male patients were observed. Additionally, we illustrated the limitations of conventional serotyping of , which was impacted by serotype switching. This study contributes to a better understanding of the spread of shigellosis within Belgium and internationally, and highlights the added value of WGS for the surveillance of this pathogen.

VL - 9 CP - 4 M3 - 10.3390/microorganisms9040767 ER - TY - JOUR T1 - Retrospective evaluation of routine whole genome sequencing of Mycobacterium tuberculosis at the Belgian National Reference Center, 2019. JF - Acta Clin Belg Y1 - 2021 A1 - Karine Soetaert A1 - Pieter-Jan Ceyssens A1 - Samira Boarbi A1 - Bert Bogaerts A1 - Thomas Delcourt A1 - Kevin Vanneste A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Alexandra Vodolazkaia A1 - Marina Mukovnikova A1 - Vanessa Mathys AB -

OBJECTIVES: Since January 2019, the Belgian National Reference Center for Mycobacteria (NRC) has switched from conventional typing to prospective whole-genome sequencing (WGS) of all submitted complex (MTB) isolates. The ISO17025 validated procedure starts with semi-automated extraction and purification of gDNA directly from the submitted MGIT tubes, without preceding subculturing. All samples are then sequenced on an Illumina MiSeq sequencer and analyzed using an in-house developed and validated bioinformatics workflow to determine the species and antimicrobial resistance. In this study, we retrospectively compare results obtained via WGS to conventional phenotypic and genotypic testing, for all Belgian MTB strains analyzed in 2019 (n = 306).

RESULTS: In all cases, the WGS-based procedure was able to identify correctly the MTB species. Compared to MGIT drug susceptibility testing (DST), the sensitivity and specificity of genetic prediction of resistance to first-line antibiotics were respectively 100 and 99% (rifampicin, RIF), 90.5 and 100% (isoniazid, INH), 100 and 98% (ethambutol, EMB) and 61.1 and 100% (pyrazinamide, PZA). The negative predictive value was above 95% for these four first-line drugs. A positive predictive value of 100% was calculated for INH and PZA, 80% for RIF and 45% for EMB.

CONCLUSIONS: Our study confirms the effectiveness of WGS for the rapid detection of complex and its drug resistance profiles for first-line drugs even when working directly on MGIT tubes, and supports the introduction of this test into the routine workflow of laboratories performing tuberculosis diagnosis.

M3 - 10.1080/17843286.2021.1999588 ER - TY - JOUR T1 - Retrospective survey of unauthorized genetically modified bacteria harbouring antimicrobial resistance genes in feed additive vitamin B2 commercialized in Belgium: Challenges and solutions JF - Food Control Y1 - 2021 A1 - Marie-Alice Fraiture A1 - Laure Joly A1 - Els Vandermassen A1 - Delvoye, Maud A1 - D. Van Geel A1 - Jean-Yves Michelet A1 - Els Van Hoeck A1 - Nathalie De Jaeger A1 - N. Papazova A1 - Nancy Roosens KW - Antimicrobial resistance genes KW - Feed additive vitamin B2 KW - food and feed safety KW - survey KW - Unauthorized genetically modified microorganisms AB -

In Belgium, an official control plan was established in 2016 to detect the potential presence of an unauthorized genetically modified (GM) Bacillus subtilis RASFF2014.1249 strain in commercialized feed additive vitamin B2 products. To this end, two real-time PCR markers specific to this unauthorized genetically modified microorganism (GMM), named UGMVit-B2 and 558, were used. In the present study, the first four-year results from 67 feed additive vitamin B2 samples from the official control are presented. It includes 5 samples positive for real-time PCR methods specific to the unauthorized GM B. subtilis RASFF2014.1249 strain and has led to the RASFF2018.2755 and RASFF2019.3216 notifications. Moreover, a retrospective study using the same feed additive vitamin B2 samples was performed, allowing to provide a first picture of GM bacterial contaminations. It consisted in a first-line screening strategy gathering available PCR-based methods targeting both the B. subtilis species, frequently used to produce vitamin B2, and a set of antimicrobial resistance (AMR) genes commonly harboured as selection marker by GM bacteria used to produce microbial fermentation products. On this basis, suspicious samples contaminated with additional unknown GM bacterial strains as well as potential health and environmental risks related to the unexpected presence of full-length AMR genes could be highlighted. In addition, the possible complementary use of additional data, like chloramphenicol presence and DNA concentration, as indicators for GMM contaminations was assessed. Based on results generated in the present study, the relevance to use the proposed first-line screening strategy supplemented by indicators in order to strengthen the current control strategy was emphasized.

VL - 119 M3 - 10.1016/j.foodcont.2020.107476 ER - TY - JOUR T1 - A shotgun metagenomics approach to detect and characterize unauthorized genetically modified microorganisms in microbial fermentation products JF - Food Chemistry: Molecular Sciences Y1 - 2021 A1 - Florence E Buytaers A1 - Marie-Alice Fraiture A1 - Bas Berbers A1 - Els Vandermassen A1 - Stefan Hoffman A1 - N. Papazova A1 - Kevin Vanneste A1 - Marchal, Kathleen A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - AMR KW - genetically modified microorganism KW - identification KW - long and short read sequencing KW - microbial fermentation products KW - shotgun metagenomics AB -

The presence of a genetically modified microorganism (GMM) or its DNA, often harboring antimicrobial resis- tance (AMR) genes, in microbial fermentation products on the market is prohibited by European regulations. GMMs are currently screened for through qPCR assays targeting AMR genes and vectors, and then confirmed by targeting known specific GM constructs/events. However, when the GMM was not previously characterized and an isolate cannot be obtained, its presence cannot be proven. We present a metagenomics approach cap- able of delivering the proof of presence of a GMM in a microbial fermentation product, with characterization based on the detection of AMR genes and vectors, species and unnatural associations in the GMM genome. In our proof‐of‐concept study, this approach was performed on a case with a previously isolated and sequenced GMM, an unresolved case for which no isolate was obtained, and a non‐GMM‐contaminated sample, all repre- sentative for the possible scenarios to occur in routine setting. Both short and long read sequencing were used. This workflow paves the way for a strategy to detect and characterize unknown GMMs by enforcement laboratories.
 

VL - 2 M3 - 10.1016/j.fochms.2021.100023 ER - TY - JOUR T1 - Strategy and Performance Evaluation of Low-Frequency Variant Calling for SARS-CoV-2 Using Targeted Deep Illumina Sequencing JF - Frontiers in Microbiology Y1 - 2021 A1 - Laura Van Poelvoorde A1 - Thomas Delcourt A1 - Wim Coucke A1 - Philippe Herman A1 - Sigrid C.J. De Keersmaecker A1 - Saelens, Xavier A1 - Nancy Roosens A1 - Kevin Vanneste KW - co-infection KW - Illumina KW - NGS KW - quasispecies KW - SARS-CoV-2 KW - variant of concern KW - wastewater surveillance AB -

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, constitutes a tremendous global health issue. Continuous monitoring of the virus has become a cornerstone to make rational decisions on implementing societal and sanitary measures to curtail the virus spread. Additionally, emerging SARS-CoV-2 variants have increased the need for genomic surveillance to detect particular strains because of their potentially increased transmissibility, pathogenicity and immune escape. Targeted SARS-CoV-2 sequencing of diagnostic and wastewater samples has been explored as an epidemiological surveillance method for the competent authorities. Currently, only the consensus genome sequence of the most abundant strain is taken into consideration for analysis, but multiple variant strains are now circulating in the population. Consequently, in diagnostic samples, potential co-infection(s) by several different variants can occur or quasispecies can develop during an infection in an individual. In wastewater samples, multiple variant strains will often be simultaneously present. Currently, quality criteria are mainly available for constructing the consensus genome sequence, and some guidelines exist for the detection of co-infections and quasispecies in diagnostic samples. The performance of detection and quantification of low-frequency variants using whole genome sequencing (WGS) of SARS-CoV-2 remains largely unknown. Here, we evaluated the detection and quantification of mutations present at low abundances using the mutations defining the SARS-CoV-2 lineage B.1.1.7 (alpha variant) as a case study. Real sequencing data were in silico modified by introducing mutations of interest into raw wild-type sequencing data, or by mixing wild-type and mutant raw sequencing data, to construct mixed samples subjected to WGS using a tiling amplicon-based targeted metagenomics approach and Illumina sequencing. As anticipated, higher variation and lower sensitivity were observed at lower coverages and allelic frequencies. We found that detection of all low-frequency variants at an abundance of 10, 5, 3, and 1%, requires at least a sequencing coverage of 250, 500, 1500, and 10,000×, respectively. Although increasing variability of estimated allelic frequencies at decreasing coverages and lower allelic frequencies was observed, its impact on reliable quantification was limited. This study provides a highly sensitive low-frequency variant detection approach, which is publicly available at https://galaxy.sciensano.be , and specific recommendations for minimum sequencing coverages to detect clade-defining mutations at certain allelic frequencies. This approach will be useful to detect and quantify low-frequency variants in both diagnostic (e.g., co-infections and quasispecies) and wastewater [e.g., multiple variants of concern (VOCs)] samples.

VL - 12 M3 - 10.3389/fmicb.2021.747458 ER - TY - JOUR T1 - Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic Evolution. JF - Curr Issues Mol Biol Y1 - 2021 A1 - Laura Van Poelvoorde A1 - Mathieu Gand A1 - Marie-Alice Fraiture A1 - Sigrid C.J. De Keersmaecker A1 - Bavo Verhaegen A1 - Koenraad Van Hoorde A1 - Ann Brigitte Cay A1 - Nadège Balmelle A1 - Philippe Herman A1 - Nancy Roosens KW - COVID-19 KW - COVID-19 Nucleic Acid Testing KW - Diagnostic Tests, Routine KW - Evolution, Molecular KW - Humans KW - RNA, Viral KW - Roc Curve KW - SARS-CoV-2 AB -

The worldwide emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has highlighted the importance of rapid and reliable diagnostic testing to prevent and control the viral transmission. However, inaccurate results may occur due to false negatives (FN) caused by polymorphisms or point mutations related to the virus evolution and compromise the accuracy of the diagnostic tests. Therefore, PCR-based SARS-CoV-2 diagnostics should be evaluated and evolve together with the rapidly increasing number of new variants appearing around the world. However, even by using a large collection of samples, laboratories are not able to test a representative collection of samples that deals with the same level of diversity that is continuously evolving worldwide. In the present study, we proposed a methodology based on an in silico and in vitro analysis. First, we used all information offered by available whole-genome sequencing data for SARS-CoV-2 for the selection of the two PCR assays targeting two different regions in the genome, and to monitor the possible impact of virus evolution on the specificity of the primers and probes of the PCR assays during and after the development of the assays. Besides this first essential in silico evaluation, a minimal set of testing was proposed to generate experimental evidence on the method performance, such as specificity, sensitivity and applicability. Therefore, a duplex reverse-transcription droplet digital PCR (RT-ddPCR) method was evaluated in silico by using 154 489 whole-genome sequences of SARS-CoV-2 strains that were representative for the circulating strains around the world. The RT-ddPCR platform was selected as it presented several advantages to detect and quantify SARS-CoV-2 RNA in clinical samples and wastewater. Next, the assays were successfully experimentally evaluated for their sensitivity and specificity. A preliminary evaluation of the applicability of the developed method was performed using both clinical and wastewater samples.

VL - 43 CP - 3 M3 - 10.3390/cimb43030134 ER - TY - JOUR T1 - A systematic review and meta-analysis of host genetic factors associated with influenza severity JF - BMC Genomics Y1 - 2021 A1 - Nina Van Goethem A1 - Célestin Danwang A1 - Nathalie Bossuyt A1 - Herman Van Oyen A1 - Nancy Roosens A1 - Robert, Annie VL - 22 CP - 1 M3 - 10.1186/s12864-021-08240-7 ER - TY - JOUR T1 - A systematic review and meta-analysis of host genetic factors associated with influenza severity JF - BMC Genomics Y1 - 2021 A1 - Nina Van Goethem A1 - Célestin Danwang A1 - Nathalie Bossuyt A1 - Herman Van Oyen A1 - Nancy Roosens A1 - Robert, Annie VL - 22 CP - 1 M3 - 10.1186/s12864-021-08240-7 ER - TY - JOUR T1 - Towards Real-Time and Affordable Strain-Level Metagenomics-Based Foodborne Outbreak Investigations Using Oxford Nanopore Sequencing Technologies JF - Frontiers in Microbiology Y1 - 2021 A1 - Florence E Buytaers A1 - Assia Saltykova A1 - Sarah Denayer A1 - Bavo Verhaegen A1 - Kevin Vanneste A1 - Nancy Roosens A1 - Piérard, Denis A1 - Marchal, Kathleen A1 - Sigrid C.J. De Keersmaecker KW - Flongle KW - food surveillance KW - Metagenomics KW - nanopore KW - outbreak KW - SNP analysis KW - STEC KW - strain-level AB -

The current routine laboratory practices to investigate food samples in case of foodborne outbreaks still rely on attempts to isolate the pathogen in order to characterize it. We present in this study a proof of concept using Shiga toxin-producing Escherichia coli spiked food samples for a strain-level metagenomics foodborne outbreak investigation method using the MinION and Flongle flow cells from Oxford Nanopore Technologies, and we compared this to Illumina short-read-based metagenomics. After 12 h of MinION sequencing, strain-level characterization could be achieved, linking the food containing a pathogen to the related human isolate of the affected patient, by means of a single-nucleotide polymorphism (SNP)-based phylogeny. The inferred strain harbored the same virulence genes as the spiked isolate and could be serotyped. This was achieved by applying a bioinformatics method on the long reads using reference-based classification. The same result could be obtained after 24-h sequencing on the more recent lower output Flongle flow cell, on an extract treated with eukaryotic host DNA removal. Moreover, an alternative approach based on in silico DNA walking allowed to obtain rapid confirmation of the presence of a putative pathogen in the food sample. The DNA fragment harboring characteristic virulence genes could be matched to the E. coli genus after sequencing only 1 h with the MinION, 1 h with the Flongle if using a host DNA removal extraction, or 5 h with the Flongle with a classical DNA extraction. This paves the way towards the use of metagenomics as a rapid, simple, one-step method for foodborne pathogen detection and for fast outbreak investigation that can be implemented in routine laboratories on samples prepared with the current standard practices.

VL - 12 M3 - 10.3389/fmicb.2021.738284 ER - TY - JOUR T1 - Validation strategy of a bioinformatics whole genome sequencing workflow for Shiga toxin-producing Escherichia coli using a reference collection extensively characterized with conventional methods JF - Microbial Genomics Y1 - 2021 A1 - Bert Bogaerts A1 - Stéphanie Nouws A1 - Bavo Verhaegen A1 - Sarah Denayer A1 - Julien Van Braekel A1 - Raf Winand A1 - Qiang Fu A1 - Florence Crombé A1 - Denis Piérard A1 - Kathleen Marchal A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker A1 - Kevin Vanneste AB -

Whole genome sequencing (WGS) enables complete characterization of bacterial pathogenic isolates at single nucleotide resolution, making it the ultimate tool for routine surveillance and outbreak investigation. The lack of standardization, and the variation regarding bioinformatics workflows and parameters, however, complicates interoperability among (inter)national laboratories. We present a validation strategy applied to a bioinformatics workflow for Illumina data that performs complete characterization of Shiga toxin-producing Escherichia coli (STEC) isolates including antimicrobial resistance prediction, virulence gene detection, serotype prediction, plasmid replicon detection and sequence typing. The workflow supports three commonly used bioinformatics approaches for the detection of genes and alleles: alignment with blast+, kmer-based read mapping with KMA, and direct read mapping with SRST2. A collection of 131 STEC isolates collected from food and human sources, extensively characterized with conventional molecular methods, was used as a validation dataset. Using a validation strategy specifically adopted to WGS, we demonstrated high performance with repeatability, reproducibility, accuracy, precision, sensitivity and specificity above 95 % for the majority of all assays. The WGS workflow is publicly available as a ‘push-button’ pipeline at https://galaxy.sciensano.be. Our validation strategy and accompanying reference dataset consisting of both conventional and WGS data can be used for characterizing the performance of various bioinformatics workflows and assays, facilitating interoperability between laboratories with different WGS and bioinformatics set-ups.

VL - 7 CP - 3 M3 - 10.1099/mgen.0.000531 ER - TY - JOUR T1 - Whole Genome Sequencing Provides an Added Value to the Investigation of Staphylococcal Food Poisoning Outbreaks JF - Frontiers in Microbiology Y1 - 2021 A1 - Stéphanie Nouws A1 - Bert Bogaerts A1 - Bavo Verhaegen A1 - Sarah Denayer A1 - Lasse Laeremans A1 - Kathleen Marchal A1 - Nancy Roosens A1 - Kevin Vanneste A1 - Sigrid C.J. De Keersmaecker KW - DNA extraction kits KW - enterotoxin gene profiling KW - outbreak investigation KW - relatedness determination KW - Staphylococcus aureus, staphylococcal food poisoning (SFP) KW - whole genome sequencing AB -

Through staphylococcal enterotoxin (SE) production, Staphylococcus aureus is a common cause of food poisoning. Detection of staphylococcal food poisoning (SFP) is mostly performed using immunoassays, which, however, only detect five of 27 SEs described to date. Polymerase chain reactions are, therefore, frequently used in complement to identify a bigger arsenal of SE at the gene level (se) but are labor-intensive. Complete se profiling of isolates from different sources, i.e., food and human cases, is, however, important to provide an indication of their potential link within foodborne outbreak investigation. In addition to complete se gene profiling, relatedness between isolates is determined with more certainty using pulsed-field gel electrophoresis, Staphylococcus protein A gene typing and other methods, but these are shown to lack resolution. We evaluated how whole genome sequencing (WGS) can offer a solution to these shortcomings. By WGS analysis of a selection of S. aureus isolates, including some belonging to a confirmed foodborne outbreak, its added value as the ultimate multiplexing method was demonstrated. In contrast to PCR-based se gene detection for which primers are sometimes shown to be non-specific, WGS enabled complete se gene profiling with high performance, provided that a database containing reference sequences for all se genes was constructed and employed. The custom compiled database and applied parameters were made publicly available in an online user-friendly interface. As an all-in-one approach with high resolution, WGS additionally allowed inferring correct isolate relationships. The different DNA extraction kits that were tested affected neither se gene profiling nor relatedness determination, which is interesting for data sharing during SFP outbreak investigation. Although confirming the production of enterotoxins remains important for SFP investigation, we delivered a proof-of-concept that WGS is a valid alternative and/or complementary tool for outbreak investigation.

VL - 12 M3 - 10.3389/fmicb.2021.750278 ER - TY - JOUR T1 - Whole-Genome Sequencing-Based Antimicrobial Resistance Characterization and Phylogenomic Investigation of 19 Multidrug-Resistant and Extended-Spectrum Beta-Lactamase-Positive Escherichia coli Strains Collected From Hospital Patients in Benin in 2019. JF - Front Microbiol Y1 - 2021 A1 - Carine Laurence Yehouenou A1 - Bert Bogaerts A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Marchal, Kathleen A1 - Edmond Tchiakpe A1 - Affolabi, Dissou A1 - Simon, Anne A1 - Francis Moise Dossou A1 - Kevin Vanneste A1 - Olivia Dalleur AB -

The increasing worldwide prevalence of extended-spectrum beta-lactamase (ESBL) producing constitutes a serious threat to global public health. Surgical site infections are associated with high morbidity and mortality rates in developing countries, fueled by the limited availability of effective antibiotics. We used whole-genome sequencing (WGS) to evaluate antimicrobial resistance and the phylogenomic relationships of 19 ESBL-positive isolates collected from surgical site infections in patients across public hospitals in Benin in 2019. Isolates were identified by MALDI-TOF mass spectrometry and phenotypically tested for susceptibility to 16 antibiotics. Core-genome multi-locus sequence typing and single-nucleotide polymorphism-based phylogenomic methods were used to investigate the relatedness between samples. The broader phylogenetic context was characterized through the inclusion of publicly available genome data. Among the 19 isolates, 13 different sequence types (STs) were observed, including ST131 ( = 2), ST38 ( = 2), ST410 ( = 2), ST405 ( = 2), ST617 ( = 2), and ST1193 ( = 2). The gene encoding ESBL resistance was found in 15 isolates (78.9%), as well as other genes associated with ESBL, such as ( = 14) and ( = 9). Additionally, we frequently observed genes encoding resistance against aminoglycosides [,  = 14], quinolones ( ,  = 4), tetracyclines [(),  = 14], sulfonamides (,  = 14), and trimethoprim (,  = 13). Nonsynonymous chromosomal mutations in the housekeeping genes and associated with resistance to fluoroquinolones were also detected in multiple isolates. Although the phylogenomic investigation did not reveal evidence of hospital-acquired transmissions, we observed two very similar strains collected from patients in different hospitals. By characterizing a set of multidrug-resistant isolates collected from a largely unexplored environment, this study highlights the added value for WGS as an effective early warning system for emerging pathogens and antimicrobial resistance.

VL - 12 M3 - 10.3389/fmicb.2021.752883 ER - TY - JOUR T1 - Whole-genome-based phylogenomic analysis of the Belgian 2016-2017 influenza A(H3N2) outbreak season allows improved surveillance. JF - Microb Genom Y1 - 2021 A1 - Laura Van Poelvoorde A1 - Bert Bogaerts A1 - Fu, Qiang A1 - Sigrid C.J. De Keersmaecker A1 - Isabelle Thomas A1 - Nina Van Goethem A1 - Steven Van Gucht A1 - Raf Winand A1 - Saelens, Xavier A1 - Nancy Roosens A1 - Kevin Vanneste KW - Belgium KW - Hemagglutinin Glycoproteins, Influenza Virus KW - Humans KW - Influenza A Virus, H3N2 Subtype KW - Influenza, Human KW - Phylogeny KW - Public Health Surveillance KW - whole genome sequencing AB -

Seasonal influenza epidemics are associated with high mortality and morbidity in the human population. Influenza surveillance is critical for providing information to national influenza programmes and for making vaccine composition predictions. Vaccination prevents viral infections, but rapid influenza evolution results in emerging mutants that differ antigenically from vaccine strains. Current influenza surveillance relies on Sanger sequencing of the haemagglutinin (HA) gene. Its classification according to World Health Organization (WHO) and European Centre for Disease Prevention and Control (ECDC) guidelines is based on combining certain genotypic amino acid mutations and phylogenetic analysis. Next-generation sequencing technologies enable a shift to whole-genome sequencing (WGS) for influenza surveillance, but this requires laboratory workflow adaptations and advanced bioinformatics workflows. In this study, 253 influenza A(H3N2) positive clinical specimens from the 2016-2017 Belgian season underwent WGS using the Illumina MiSeq system. HA-based classification according to WHO/ECDC guidelines did not allow classification of all samples. A new approach, considering the whole genome, was investigated based on using powerful phylogenomic tools including beast and Nextstrain, which substantially improved phylogenetic classification. Moreover, Bayesian inference via beast facilitated reassortment detection by both manual inspection and computational methods, detecting intra-subtype reassortants at an estimated rate of 15 %. Real-time analysis (i.e. as an outbreak is ongoing) via Nextstrain allowed positioning of the Belgian isolates into the globally circulating context. Finally, integration of patient data with phylogenetic groups and reassortment status allowed detection of several associations that would have been missed when solely considering HA, such as hospitalized patients being more likely to be infected with A(H3N2) reassortants, and the possibility to link several phylogenetic groups to disease severity indicators could be relevant for epidemiological monitoring. Our study demonstrates that WGS offers multiple advantages for influenza monitoring in (inter)national influenza surveillance, and proposes an improved methodology. This allows leveraging all information contained in influenza genomes, and allows for more accurate genetic characterization and reassortment detection.

VL - 7 CP - 9 M3 - 10.1099/mgen.0.000643 ER - TY - JOUR T1 - The Benefits of Whole Genome Sequencing for Foodborne Outbreak Investigation from the Perspective of a National Reference Laboratory in a Smaller Country. JF - Foods Y1 - 2020 A1 - Stéphanie Nouws A1 - Bert Bogaerts A1 - Bavo Verhaegen A1 - Sarah Denayer A1 - Florence Crombé A1 - Klara De Rauw A1 - Denis Piérard A1 - Kathleen Marchal A1 - Kevin Vanneste A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - Food Safety KW - foodborne outbreak investigation KW - Shiga toxin-producing Escherichia coli KW - STEC KW - Surveillance KW - WGS KW - whole genome sequencing AB -

Gradually, conventional methods for foodborne pathogen typing are replaced by whole genome sequencing (WGS). Despite studies describing the overall benefits, National Reference Laboratories of smaller countries often show slower uptake of WGS, mainly because of significant investments required to generate and analyze data of a limited amount of samples. To facilitate this process and incite policy makers to support its implementation, a Shiga toxin-producing (STEC) O157:H7 (+, +, +) outbreak (2012) and a STEC O157:H7 (+, +) outbreak (2013) were retrospectively analyzed using WGS and compared with their conventional investigations. The corresponding results were obtained, with WGS delivering even more information, e.g., on virulence and antimicrobial resistance genotypes. Besides a universal, all-in-one workflow with less hands-on-time (five versus seven actual working days for WGS versus conventional), WGS-based cgMLST-typing demonstrated increased resolution. This enabled an accurate cluster definition, which remained unsolved for the 2013 outbreak, partly due to scarce epidemiological linking with the suspect source. Moreover, it allowed detecting two and one earlier circulating STEC O157:H7 (+, +, +) and STEC O157:H7 (+, +) strains as closely related to the 2012 and 2013 outbreaks, respectively, which might have further directed epidemiological investigation initially. Although some bottlenecks concerning centralized data-sharing, sampling strategies, and perceived costs should be considered, we delivered a proof-of-concept that even in smaller countries, WGS offers benefits for outbreak investigation, if a sufficient budget is available to ensure its implementation in surveillance. Indeed, applying a database with background isolates is critical in interpreting isolate relationships to outbreaks, and leveraging the true benefit of WGS in outbreak investigation and/or prevention.

VL - 9 CP - 8 M3 - 10.3390/foods9081030 ER - TY - JOUR T1 - Combining short and long read sequencing to characterize antimicrobial resistance genes on plasmids applied to an unauthorized genetically modified Bacillus JF - Scientific Reports Y1 - 2020 A1 - Bas Berbers A1 - Assia Saltykova A1 - Cristina Garcia-Graells A1 - Philipp, Patrick A1 - Fabrice Arella A1 - Marchal, Kathleen A1 - Raf Winand A1 - Kevin Vanneste A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - AMR KW - GMM KW - long and short read sequencing KW - NGS KW - Plasmids AB -

Antimicrobial resistance (AMR) is a major public health threat. Plasmids are able to transfer AMR genes among bacterial isolates. Whole genome sequencing (WGS) is a powerful tool to monitor AMR determinants. However, plasmids are difficult to reconstruct from WGS data. This study aimed to improve the characterization, including the localization of AMR genes using short and long read WGS strategies. We used a genetically modified (GM) Bacillus subtilis isolated as unexpected contamination in a feed additive, and therefore considered unauthorized (RASFF 2014.1249), as a case study. In GM organisms, AMR genes are used as selection markers. Because of the concern of spread of these AMR genes when present on mobile genetic elements, it is crucial to characterize their location. Our approach resulted in an assembly of one chromosome and one plasmid, each with several AMR determinants of which five are against critically important antibiotics. Interestingly, we found several plasmids, containing AMR genes, integrated in the chromosome in a repetitive region of at least 53 kb. Our findings would have been impossible using short reads only. We illustrated the added value of long read sequencing in addressing the challenges of plasmid reconstruction within the context of evaluating the risk of AMR spread.

VL - 10 CP - 1 M3 - 10.1038/s41598-020-61158-0 ER - TY - JOUR T1 - Detection strategy targeting a chloramphenicol resistance gene from genetically modified bacteria in food and feed products JF - Food Control Y1 - 2020 A1 - Marie-Alice Fraiture A1 - Marie Deckers A1 - N. Papazova A1 - Nancy Roosens KW - Chloramphenicol resistance gene KW - Food and feed microbial fermentation products KW - Genetically modified microorganisms KW - PCR-Based detection AB -

Genetically modified microorganisms (GMM), harbouring commonly antimicrobial resistance (AMR) genes as selection markers, are frequently used to produce food and feed enzymes, additives and flavourings. Such commercialized microbial fermentation products should not contain GMM, or associated recombinant DNA. Although the use of AMR genes gives rise to public health and environmental concerns regarding their potential acquisitions by pathogens and gut microbiota, no method targeting AMR genes harboured by such GMM is currently available for the enforcement laboratories. In reason of the increasing interest of the competent authorities to be able to assess the potential risks related to the presence of these AMR genes in microbial fermentation products, we propose therefore for the first time a PCR-based strategy easily implementable in enforcement laboratories. This strategy targets a chloramphenicol resistance gene, highlighted by the patent analysis performed in this study as being harboured by a noteworthy part of GMM producing microbial fermentation products from the food and feed industry. First, the potential presence of the AMR gene is detected by real-time PCR. Next, its full-length is evaluated by a nested-PCR amplifying a large fragment of its sequence to determine the risks of likely AMR gene acquisition. This strategy allows thus to support the competent authorities regarding the measures to be taken in case of unexpected DNA contaminations from such GMM in commercialized microbial fermentation products.

VL - 108 M3 - 10.1016/j.foodcont.2019.106873 ER - TY - JOUR T1 - Development of an NGS-Based Workflow for Improved Monitoring of Circulating Plasmids in Support of Risk Assessment of Antimicrobial Resistance Gene Dissemination. JF - Antibiotics (Basel) Y1 - 2020 A1 - Bas Berbers A1 - Pieter-Jan Ceyssens A1 - Bogaerts, Pierre A1 - Kevin Vanneste A1 - Nancy Roosens A1 - Marchal, Kathleen A1 - Sigrid C.J. De Keersmaecker KW - Antimicrobial resistance KW - MinION KW - Plasmid sequencing AB -

Antimicrobial resistance (AMR) is one of the most prominent public health threats. AMR genes localized on plasmids can be easily transferred between bacterial isolates by horizontal gene transfer, thereby contributing to the spread of AMR. Next-generation sequencing (NGS) technologies are ideal for the detection of AMR genes; however, reliable reconstruction of plasmids is still a challenge due to large repetitive regions. This study proposes a workflow to reconstruct plasmids with NGS data in view of AMR gene localization, i.e., chromosomal or on a plasmid. Whole-genome and plasmid DNA extraction methods were compared, as were assemblies consisting of short reads (Illumina MiSeq), long reads (Oxford Nanopore Technologies) and a combination of both (hybrid). Furthermore, the added value of conjugation of a plasmid to a known host was evaluated. As a case study, an isolate harboring a large, low-copy -carrying plasmid (>200 kb) was used. Hybrid assemblies of NGS data obtained from whole-genome DNA extractions of the original isolates resulted in the most complete reconstruction of plasmids. The optimal workflow was successfully applied to multidrug-resistant Kentucky isolates, where the transfer of an ESBL-gene-containing fragment from a plasmid to the chromosome was detected. This study highlights a strategy including wet and dry lab parameters that allows accurate plasmid reconstruction, which will contribute to an improved monitoring of circulating plasmids and the assessment of their risk of transfer.

VL - 9 CP - 8 M3 - 10.3390/antibiotics9080503 ER - TY - JOUR T1 - First detection of a plasmid located carbapenem resistant blaVIM-1 gene in E. coli isolated from meat products at retail in Belgium in 2015 JF - International Journal of Food Microbiology Y1 - 2020 A1 - Cristina Garcia-Graells A1 - Bas Berbers A1 - Bavo Verhaegen A1 - Kevin Vanneste A1 - Marchal, Kathleen A1 - Nancy Roosens A1 - N Botteldoorn A1 - Sigrid C.J. De Keersmaecker VL - 324 M3 - 10.1016/j.ijfoodmicro.2020.108624 ER - TY - BOOK T1 - Food Chemistry, Function and AnalysisDNA Techniques to Verify Food Authenticity CHAPTER 8. GMO Detection and Identification Using Next-generation Sequencing Y1 - 2020 A1 - Marie-Alice Fraiture A1 - N. Papazova A1 - Kevin Vanneste A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens KW - GMO Detection and Identification Using Next-generation AB -

ext-generation sequencing (NGS) technologies are being increasingly evaluated for their feasibility in supporting the current genetically modified organism (GMO) routine detection systems for unauthorized GMO. Depending upon the specific samples under investigation, two categories of NGS approaches can be applied. The whole-genome sequencing approach, on the one hand, requires no prior knowledge, and is suitable for the characterization of samples containing an isolated single GMO. The targeted approach, on the other hand, requires upstream enrichment of a priori known sequences of interest, and allows simultaneous identification of several GMOs, even when present at low concentrations. The application of these NGS approaches to biotech organisms created by genome editing techniques, which are currently not included in the scope of European Union (EU) legislation, is also discussed.

PB - Royal Society of Chemistry CY - Cambridge SN - 978-1-78801-178-5 M3 - 10.1039/9781788016025-00096 ER - TY - JOUR T1 - Gender‐dependent association between exhaled nitric oxide and the CC16 38AA genotype in young school children JF - Immunity, Inflammation and Disease Y1 - 2020 A1 - S. Nauwelaerts A1 - Nancy Roosens A1 - Koen De Cremer A1 - Bernard, Alfred A1 - Sigrid C.J. De Keersmaecker AB -

Background

Studies that investigated the association between the CC16 A38G polymorphism and the risk of asthma yielded conflicting results. The aim of this study among schoolchildren was to assess the relationships of CC16 A38G polymorphism with aeroallergen sensitization and fractional exhaled nitric oxide (FeNO), two outcomes predicting asthma later in life.

Methods

The study included 139 children (72 boys), median age of 7.7. Information on each child's health, lifestyle, and environment was collected through a questionnaire completed by their parents. CC16 genotypes were determined using urinary DNA. We measured FeNO, the CC16 protein in urine and nasal lavage fluid and aeroallergen‐specific immunoglobulin E in nasal mucosa fluid.

Results

Children with the homozygous mutant CC16 38AA genotype had higher odds of increased FeNO (>30 ppb) compared with their peers with the wild‐type genotype 38GG (OR, 9.85; 95% CI, 2.09‐46.4; P = .004). This association was female gender specific (P = .002) not being observed in boys (P = .40). It was also independent of allergic sensitization, which yet emerged as the strongest predictor of FeNO along with the use of bleach for house cleaning. Children with the CC16 38AA genotype had lower covariates‐adjusted urinary CC16 levels than those with 38GG (median, μg/L, 1.17 vs 2.08, P = .02).

Conclusion

Our study suggests that the CC16 38AA allele promotes airway inflammation as measured by FeNO through a gender‐dependent association. Deficient levels of CC16 in the deep lung, measured noninvasively in urine, as a possible proxy for serum CC16, might underlie this promoting effect.

M3 - 10.1002/iid3.332 ER - TY - JOUR T1 - A genoserotyping system for a fast and objective identification of Salmonella serotypes commonly isolated from poultry and pork food sectors in Belgium. JF - Food Microbiol Y1 - 2020 A1 - Mathieu Gand A1 - Wesley Mattheus A1 - Nancy Roosens A1 - Katelijne Dierick A1 - Marchal, Kathleen A1 - Sophie Bertrand A1 - Sigrid C.J. De Keersmaecker KW - Salmonella; Genoserotyping; Luminex; Decision support system; Food safety AB -

Humans are mostly contaminated by Salmonella through the consumption of pork- and poultry-derived food products. Therefore, a strict monitoring of Salmonella serotypes in food-producing animals is needed to limit the transmission of the pathogen to humans. Additionally, Salmonella can lead to economic loss in the food sector. Previously, a genoserotyping method using the MOL-PCR and Luminex technology was developed for the identification of the 6 Salmonella serotypes, and their variants, subjected to an official control in the Belgian food sector. In this study, 3 additional assays using the same technology were developed for the rapid and cost-effective detection of 13 dangerous highly invasive serotypes or other serotypes frequently isolated from the Belgian poultry and pork sector, i.e. Agona, Anatum, Brandenburg, Choleraesuis, Derby, Enteritidis vaccine strains, Gallinarum var. Gallinarum/Pullorum, Livingstone, Mbandaka, Minnesota, Ohio, Rissen and Senftenberg. Moreover, the previously developed first MOL-PCR assay was improved for S. Paratyphi B and serogroup O:3 detection. Finally, a Decision Support System hosted by a web application was created for an automatic and objective interpretation of the Luminex raw data. The 3 new assays and the modifications of the first assay were validated with a 100% accuracy, using 553 Salmonella and non-Salmonella strains in total.

VL - 91 M3 - 10.1016/j.fm.2020.103534 ER - TY - JOUR T1 - Identification of an unauthorized genetically modified bacteria in food enzyme through whole-genome sequencing. JF - Sci Rep Y1 - 2020 A1 - Marie-Alice Fraiture A1 - Bert Bogaerts A1 - Raf Winand A1 - Marie Deckers A1 - N. Papazova A1 - Kevin Vanneste A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens KW - Drug Resistance, Bacterial KW - Food Microbiology KW - Genetic Vectors KW - Microorganisms, Genetically-Modified KW - Peptide Hydrolases KW - whole genome sequencing AB -

Recently, the unexpected presence of a viable unauthorized genetically modified bacterium in a commercialized food enzyme (protease) product originating from a microbial fermentation process has been notified at the European level (RASFF 2019.3332). This finding was made possible thanks to the use of the next-generation sequencing technology, as reported in this study. Whole-genome sequencing was used to characterize the genetic modification comprising a sequence from the pUB110 shuttle vector (GenBank: M19465.1), harbouring antimicrobial resistance genes conferring a resistance to kanamycine, neomycin and bleomycin, flanked on each side by a sequence coding for a protease (GenBank: WP_032874795.1). In addition, based on these data, two real-time PCR methods, that can be used by enforcement laboratories, specific to this unauthorized genetically modified bacterium were developed and validated. The present study emphasizes the key role that whole-genome sequencing can take for detection of unknown and unauthorized genetically modified microorganisms in commercialized microbial fermentation products intended for the food and feed chain. Moreover, current issues encountered by the Competent Authorities and enforcement laboratories with such unexpected contaminations and the importance of performing official controls were highlighted.

VL - 10 CP - 1 M3 - 10.1038/s41598-020-63987-5 ER - TY - JOUR T1 - Impact of DNA extraction on whole genome sequencing analysis for characterization and relatedness of Shiga toxin-producing Escherichia coli isolates JF - Scientific Reports Y1 - 2020 A1 - Stéphanie Nouws A1 - Bert Bogaerts A1 - Bavo Verhaegen A1 - Sarah Denayer A1 - Piérard, Denis A1 - Marchal, Kathleen A1 - Nancy Roosens A1 - Kevin Vanneste A1 - Sigrid C.J. De Keersmaecker KW - DNA extraction kits KW - outbreak investigation KW - Shiga toxin producing Escherichia coli (STEC) KW - Whole genome sequencing (WGS) AB -

Whole genome sequencing (WGS) has proven to be the ultimate tool for bacterial isolate characterization and relatedness determination. However, standardized and harmonized workflows, e.g. for DNA extraction, are required to ensure robust and exchangeable WGS data. Data sharing between (inter)national laboratories is essential to support foodborne pathogen control, including outbreak investigation. This study evaluated eight commercial DNA preparation kits for their potential influence on: (i) DNA quality for Nextera XT library preparation; (ii) MiSeq sequencing (data quality, read mapping against plasmid and chromosome references); and (iii) WGS data analysis, i.e. isolate characterization (serotyping, virulence and antimicrobial resistance genotyping) and phylogenetic relatedness (core genome multilocus sequence typing and single nucleotide polymorphism analysis). Shiga toxin-producing Escherichia coli (STEC) was selected as a case study. Overall, data quality and inferred phylogenetic relationships between isolates were not affected by the DNA extraction kit choice, irrespective of the presence of confounding factors such as EDTA in DNA solution buffers. Nevertheless, completeness of STEC characterization was, although not substantially, influenced by the plasmid extraction performance of the kits, especially when using Nextera XT library preparation. This study contributes to addressing the WGS challenges of standardizing protocols to support data portability and to enable full exploitation of its potential.

VL - 10 CP - 1 M3 - 10.1038/s41598-020-71207-3 ER - TY - JOUR T1 - Impact of DNA extraction on whole genome sequencing analysis for characterization and relatedness of Shiga toxin-producing Escherichia coli isolates. JF - Sci Rep Y1 - 2020 A1 - Stéphanie Nouws A1 - Bert Bogaerts A1 - Bavo Verhaegen A1 - Sarah Denayer A1 - Piérard, Denis A1 - Marchal, Kathleen A1 - Nancy Roosens A1 - Kevin Vanneste A1 - Sigrid C.J. De Keersmaecker KW - NGS KW - pathogenic E. coli KW - STEC KW - WGS AB -

Whole genome sequencing (WGS) has proven to be the ultimate tool for bacterial isolate characterization and relatedness determination. However, standardized and harmonized workflows, e.g. for DNA extraction, are required to ensure robust and exchangeable WGS data. Data sharing between (inter)national laboratories is essential to support foodborne pathogen control, including outbreak investigation. This study evaluated eight commercial DNA preparation kits for their potential influence on: (i) DNA quality for Nextera XT library preparation; (ii) MiSeq sequencing (data quality, read mapping against plasmid and chromosome references); and (iii) WGS data analysis, i.e. isolate characterization (serotyping, virulence and antimicrobial resistance genotyping) and phylogenetic relatedness (core genome multilocus sequence typing and single nucleotide polymorphism analysis). Shiga toxin-producing Escherichia coli (STEC) was selected as a case study. Overall, data quality and inferred phylogenetic relationships between isolates were not affected by the DNA extraction kit choice, irrespective of the presence of confounding factors such as EDTA in DNA solution buffers. Nevertheless, completeness of STEC characterization was, although not substantially, influenced by the plasmid extraction performance of the kits, especially when using Nextera XT library preparation. This study contributes to addressing the WGS challenges of standardizing protocols to support data portability and to enable full exploitation of its potential.

VL - 10 CP - 1 M3 - 10.1038/s41598-020-71207-3 ER - TY - JOUR T1 - Isolation of Drug-Resistant Gallibacterium anatis from Calves with Unresponsive Bronchopneumonia, Belgium. JF - Emerg Infect Dis Y1 - 2020 A1 - Van Driessche, Laura A1 - Kevin Vanneste A1 - Bert Bogaerts A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Haesebrouck, Freddy A1 - Lieze De Cremer A1 - Deprez, Piet A1 - Pardon, Bart A1 - Boyen, Filip KW - Animals KW - Belgium KW - Bronchopneumonia KW - Cattle KW - Cattle Diseases KW - Drug Resistance, Microbial KW - Pasteurellaceae AB -

Gallibacterium anatis is an opportunistic pathogen, previously associated with deaths in poultry, domestic birds, and occasionally humans. We obtained G. anatis isolates from bronchoalveolar lavage samples of 10 calves with bronchopneumonia unresponsive to antimicrobial therapy. Collected isolates were multidrug-resistant to extensively drug-resistant, exhibiting resistance against 5-7 classes of antimicrobial drugs. Whole-genome sequencing revealed 24 different antimicrobial-resistance determinants, including genes not previously described in the Gallibacterium genus or even the Pasteurellaceae family, such as aadA23, bla, tet(Y), and qnrD1. Some resistance genes were closely linked in resistance gene cassettes with either transposases in close proximity or situated on putative mobile elements or predicted plasmids. Single-nucleotide polymorphism genotyping revealed large genetic variation between the G. anatis isolates, including isolates retrieved from the same farm. G. anatis might play a hitherto unrecognized role as a respiratory pathogen and resistance gene reservoir in cattle and has unknown zoonotic potential.

VL - 26 CP - 4 M3 - 10.3201/eid2604.190962 ER - TY - JOUR T1 - A multiplex oligonucleotide ligation-PCR method for the genoserotyping of common Salmonella using a liquid bead suspension assay JF - Food Microbiology Y1 - 2020 A1 - Mathieu Gand A1 - Wesley Mattheus A1 - Nancy Roosens A1 - Katelijne Dierick A1 - Marchal, Kathleen A1 - Sigrid C.J. De Keersmaecker A1 - Sophie Bertrand VL - 87 M3 - 10.1016/j.fm.2019.103394 ER - TY - JOUR T1 - A new multiplex RT-qPCR method for the simultaneous detection and discrimination of Zika and chikungunya viruses JF - International Journal of Infectious Diseases Y1 - 2020 A1 - Sylvia Broeders A1 - Linda Garlant A1 - Marie-Alice Fraiture A1 - Els Vandermassen A1 - Vanessa Suin A1 - Jessica Vanhomwegen A1 - Myrielle Dupont-Rouzeyrol A1 - Dominique Rousset A1 - Steven Van Gucht A1 - Nancy Roosens KW - Chikungunya virus KW - discrimination KW - identification KW - multiplex KW - RT-qPCR KW - Zika virus AB -

Objective

The re-emergence and spread of tropical viruses to new areas has raised a wave of concern worldwide. In order to treat patients at an early stage and prevent the diffusion of an outbreak, early diagnosis, and therefore fast and adequate detection, is needed. To this end, a multiplex reverse transcription real-time polymerase chain reaction TaqMan method was designed to detect Zika (ZIKV) and chikungunya (CHIKV) viruses simultaneously.

Methods

Two methods targeting different genome segments were selected from the literature for each virus. These were adapted for high genome coverage and combined in a four-plex assay that was thoroughly validated in-house. The SCREENED tool was used to evaluate the sequence coverage of the method.

Results

The full validation approach showed that the new four-plex method allows the specific and sensitive identification and discrimination of ZIKV and CHIKV in routine samples. The combination of two targets per virus allowing almost 100% coverage of about 500 genomes is shown for the first time.

Conclusions

PCR is a reliable user-friendly technique that can be applied in remote areas. Such multiplex methods enable early and efficient diagnosis, leading to rapid treatment and effective confinement in outbreak cases. They may also serve as an aid for surveillance, and the full validation permits easy method-transfer allowing worldwide harmonization.

VL - 92 M3 - 10.1016/j.ijid.2019.12.028 ER - TY - JOUR T1 - Next-Generation Sequencing: An Eye-Opener for the Surveillance of Antiviral Resistance in Influenza JF - Trends in Biotechnology Y1 - 2020 A1 - Laura Van Poelvoorde A1 - Xavier Saelens A1 - Isabelle Thomas A1 - Nancy Roosens KW - antiviral resistance KW - INFLUENZA KW - Next-generation sequencing KW - Surveillance AB -

Next-generation sequencing (NGS) can enable a more effective response to a wide range of communicable disease threats, such as influenza, which is one of the leading causes of human morbidity and mortality worldwide. After vaccination, antivirals are the second line of defense against influenza. The use of currently available antivirals can lead to antiviral resistance mutations in the entire influenza genome. Therefore, the methods to detect these mutations should be developed and implemented. In this Opinion, we assess how NGS could be implemented to detect drug resistance mutations in clinical influenza virus isolates.

VL - 38 CP - 4 M3 - 10.1016/j.tibtech.2019.09.009 ER - TY - JOUR T1 - NGS for (Hemato-) Oncology in Belgium: Evaluation of Laboratory Performance and Feasibility of a National External Quality Assessment Program JF - Cancers Y1 - 2020 A1 - Thomas Delcourt A1 - Kevin Vanneste A1 - Mohamed Rida Soumali A1 - Wim Coucke A1 - V Ghislain A1 - Aline Hébrant A1 - Els Van Valckenborgh A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - P Van de Walle A1 - Marc Van den Bulcke A1 - Aline Antoniou KW - next-generation sequencing; hemato-oncology; oncology; external quality assessment; cancer AB -

Next-generation sequencing (NGS) is being integrated into routine clinical practice in the field of (hemato-) oncology to search for variants with diagnostic, prognostic, or therapeutic value at potentially low allelic frequencies. The complex sequencing workflows used require careful validation and continuous quality control. Participation in external quality assessments (EQA) helps laboratories evaluate their performance and guarantee the validity of tests results with the ultimate goal of ensuring high-quality patient care. Here, we describe three benchmarking trials performed during the period 2017–2018 aiming firstly at establishing the state-of-the-art and secondly setting up a NGS-specific EQA program at the national level in the field of clinical (hemato-) oncology in Belgium. DNAsamples derived from cell line mixes and artificially mutated cell lines, designed to carry variants of clinical relevance occurring in solid tumors, hematological malignancies, and BRCA1/BRCA2 genes, were sent to Belgian human genetics, anatomic pathology, and clinical biology laboratories, to be processed following routine practices, together with surveys covering technical aspects of the NGS workflows. Despite the wide variety of platforms and workflows currently applied in routine clinical practice, performance was satisfactory, since participating laboratories identified the targeted variants with success rates ranging between 93.06% and 97.63% depending on the benchmark, and few false negative or repeatability issues were identified. However, variant reporting and interpretation varied, underlining the need for further standardization. Our approach showcases the feasibility of developing and implementing EQA for routine clinical practice in the field of (hemato-) oncology, while highlighting the challenges faced.

VL - 12 CP - 11 M3 - 10.3390/cancers12113180 ER - TY - JOUR T1 - Perceived utility and feasibility of pathogen genomics for public health practice: a survey among public health professionals working in the field of infectious diseases, Belgium, 2019 JF - BMC Public Health Y1 - 2020 A1 - Nina Van Goethem A1 - Marc J Struelens A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Annie Robert A1 - Sophie Quoilin A1 - Herman Van Oyen A1 - Brecht Devleesschauwer KW - Next-generation sequencing KW - pathogen genomics KW - Public Health Practice KW - survey KW - whole-genome sequencing AB -

Background

Pathogen genomics is increasingly being translated from the research setting into the activities of public health professionals operating at different levels. This survey aims to appraise the literacy level and gather the opinions of public health experts and allied professionals working in the field of infectious diseases in Belgium concerning the implementation of next-generation sequencing (NGS) in public health practice.

Methods

In May 2019, Belgian public health and healthcare professionals were invited to complete an online survey containing eight main topics including background questions, general attitude towards pathogen genomics for public health practice and main concerns, genomic literacy, current and planned NGS activities, place of NGS in diagnostic microbiology pathways, data sharing obstacles, end-user requirements, and key drivers for the implementation of NGS. Descriptive statistics were used to report on the frequency distribution of multiple choice responses whereas thematic analysis was used to analyze free text responses. A multivariable logistic regression model was constructed to identify important predictors for a positive attitude towards the implementation of pathogen genomics in public health practice.

Results

146 out of the 753 invited public health professionals completed the survey. 63% of respondents indicated that public health agencies should be using genomics to understand and control infectious diseases. Having a high level of expertise in the field of pathogen genomics was the strongest predictor of a positive attitude (OR = 4.04, 95% CI = 1.11 – 17.23). A significantly higher proportion of data providers indicated to have followed training in the field of pathogen genomics compared to data end-users (p < 0.001). Overall, 79% of participants expressed interest in receiving further training. Main concerns were related to the cost of sequencing technologies, data sharing, data integration, interdisciplinary working, and bioinformatics expertise.

Conclusions

Belgian health professionals expressed favorable views about implementation of pathogen genomics in their work activities related to infectious disease surveillance and control. They expressed the need for suitable training initiatives to strengthen their competences in the field. Their perception of the utility and feasibility of pathogen genomics for public health purposes will be a key driver for its further implementation.

VL - 20 CP - 1 M3 - 10.1186/s12889-020-09428-4 ER - TY - JOUR T1 - Perceived utility and feasibility of pathogen genomics for public health practice: a survey among public health professionals working in the field of infectious diseases, Belgium, 2019 JF - BMC Public Health Y1 - 2020 A1 - Nina Van Goethem A1 - M. J. Struelens A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - A. Robert A1 - Sophie Quoilin A1 - Herman Van Oyen A1 - Brecht Devleesschauwer VL - 20 CP - 1 M3 - 10.1186/s12889-020-09428-4 ER - TY - JOUR T1 - A Practical Method to Implement Strain-Level Metagenomics-Based Foodborne Outbreak Investigation and Source Tracking in Routine. JF - Microorganisms Y1 - 2020 A1 - Florence E Buytaers A1 - Assia Saltykova A1 - Sarah Denayer A1 - Bavo Verhaegen A1 - Kevin Vanneste A1 - Nancy Roosens A1 - Piérard, Denis A1 - Marchal, Kathleen A1 - Sigrid C.J. De Keersmaecker KW - food surveillance KW - Metagenomics KW - outbreak KW - SNP analysis KW - STEC KW - whole genome AB -

The management of a foodborne outbreak depends on the rapid and accurate identification of the responsible food source. Conventional methods based on isolation of the pathogen from the food matrix and target-specific real-time polymerase chain reactions (qPCRs) are used in routine. In recent years, the use of whole genome sequencing (WGS) of bacterial isolates has proven its value to collect relevant information for strain characterization as well as tracing the origin of the contamination by linking the food isolate with the patient's isolate with high resolution. However, the isolation of a bacterial pathogen from food matrices is often time-consuming and not always successful. Therefore, we aimed to improve outbreak investigation by developing a method that can be implemented in reference laboratories to characterize the pathogen in the food vehicle without its prior isolation and link it back to human cases. We tested and validated a shotgun metagenomics approach by spiking food pathogens in specific food matrices using the Shiga toxin-producing (STEC) as a case study. Different DNA extraction kits and enrichment procedures were investigated to obtain the most practical workflow. We demonstrated the feasibility of shotgun metagenomics to obtain the same information as in ISO/TS 13136:2012 and WGS of the isolate in parallel by inferring the genome of the contaminant and characterizing it in a shorter timeframe. This was achieved in food samples containing different strains, including a combination of different STEC strains. For the first time, we also managed to link individual strains from a food product to isolates from human cases, demonstrating the power of shotgun metagenomics for rapid outbreak investigation and source tracking.

VL - 8 CP - 8 M3 - 10.3390/microorganisms8081191 ER - TY - JOUR T1 - Screening strategy targeting the presence of food enzyme-producing fungi in food enzyme preparations JF - Food Control Y1 - 2020 A1 - Marie Deckers A1 - Kevin Vanneste A1 - Raf Winand A1 - Marijke Hendrickx A1 - Pierre Becker A1 - Sigrid C.J. De Keersmaecker A1 - Deforce, Dieter A1 - Marie-Alice Fraiture A1 - Nancy Roosens KW - Food enzymes KW - Fungi VL - 117 M3 - 10.1016/j.foodcont.2020.107295 ER - TY - JOUR T1 - Selection of a Noninvasive Source of Human DNA Envisaging Genotyping Assays in Epidemiological Studies: Urine or Saliva? JF - Journal of Biomolecular Techniques : JBT Y1 - 2020 A1 - S. Nauwelaerts A1 - D. Van Geel A1 - Delvoye, Maud A1 - Koen De Cremer A1 - Bernard, Alfred A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - Biomarkers KW - children. KW - genetic epidemiology KW - Genotype KW - Real-time PCR AB -

Genetic epidemiology requires an appropriate approach to measure genetic variation within the population. The aim of this study was to evaluate the characteristics and genotyping results of DNA extracted from 2 human DNA sources, selected for their rapid and noninvasive sampling, and the use of simple and standardized protocols that are essential for large-scale epidemiologic studies. Saliva and urine samples were collected at the same day from 20 subjects aged 9-10 yr. Genomic DNA was extracted using commercial kits. Quantitative and qualitative evaluation was done by assessing the yield, the purity, and integrity of the extracted DNA. As a proof-of-concept, genotyping was performed targeting CC16 A38G and uteroglobin-related protein 1 (UGRP1)-112G/A. Saliva was found to provide the highest yield and concentration of total DNA extracted. Salivary DNA showed higher purity and a significantly less degraded state compared to urinary DNA. Consequently, the salivary DNA gave better genotyping results than urinary DNA. Therefore, if the choice exists, saliva is the preferred noninvasive matrix for genotyping purposes in large-scale genetic epidemiologic studies. Only in particular cases using urine could nevertheless be considered useful, although specific limitations need to be taken into account.

VL - 31 CP - 1 M3 - 10.7171/jbt.20-3101-004 ER - TY - JOUR T1 - Strain-Level Metagenomic Data Analysis of Enriched In Vitro and In Silico Spiked Food Samples: Paving the Way towards a Culture-Free Foodborne Outbreak Investigation Using STEC as a Case Study. JF - Int J Mol Sci Y1 - 2020 A1 - Assia Saltykova A1 - Florence E Buytaers A1 - Sarah Denayer A1 - Bavo Verhaegen A1 - Piérard, Denis A1 - Nancy Roosens A1 - Marchal, Kathleen A1 - Sigrid C.J. De Keersmaecker KW - foodborne outbreak investigation KW - public health KW - strain-level metagenomics AB -

Culture-independent diagnostics, such as metagenomic shotgun sequencing of food samples, could not only reduce the turnaround time of samples in an outbreak investigation, but also allow the detection of multi-species and multi-strain outbreaks. For successful foodborne outbreak investigation using a metagenomic approach, it is, however, necessary to bioinformatically separate the genomes of individual strains, including strains belonging to the same species, present in a microbial community, which has up until now not been demonstrated for this application. The current work shows the feasibility of strain-level metagenomics of enriched food matrix samples making use of data analysis tools that classify reads against a sequence database. It includes a brief comparison of two database-based read classification tools, Sigma and Sparse, using a mock community obtained by in vitro spiking minced meat with a Shiga toxin-producing (STEC) isolate originating from a described outbreak. The more optimal tool Sigma was further evaluated using in silico simulated metagenomic data to explore the possibilities and limitations of this data analysis approach. The performed analysis allowed us to link the pathogenic strains from food samples to human isolates previously collected during the same outbreak, demonstrating that the metagenomic approach could be applied for the rapid source tracking of foodborne outbreaks. To our knowledge, this is the first study demonstrating a data analysis approach for detailed characterization and phylogenetic placement of multiple bacterial strains of one species from shotgun metagenomic WGS data of an enriched food sample.

VL - 21 CP - 16 M3 - 10.3390/ijms21165688 ER - TY - JOUR T1 - Strategy for the identification of micro-organisms producing food and feed products: Bacteria producing food enzymes as study case JF - Food Chemistry Y1 - 2020 A1 - Marie Deckers A1 - Kevin Vanneste A1 - Raf Winand A1 - Sigrid C.J. De Keersmaecker A1 - Sarah Denayer A1 - Marc Heyndrickx A1 - Dieter Deforce A1 - Marie-Alice Fraiture A1 - Nancy Roosens KW - 16S-rRNA gene sequencing KW - bacteria KW - Food enzymes KW - identification KW - PCR technology KW - Producing organisms KW - SCREENING AB -

Recent European regulations require safety assessments of food enzymes (FE) before their commercialization. FE are mainly produced by micro-organisms, whose viable strains nor associated DNA can be present in the final products. Currently, no strategy targeting such impurities exists in enforcement laboratories. Therefore, a generic strategy of first line screening was developed to detect and identify, through PCR amplification and sequencing of the 16S-rRNA gene, the potential presence of FE producing bacteria in FE preparations. First, the specificity was verified using all microbial species reported to produce FE. Second, an in-house database, with 16S reference sequences from bacteria producing FE, was constructed for their fast identification through blast analysis. Third, the sensitivity was assessed on a spiked FE preparation. Finally, the applicability was verified using commercial FE preparations. Using straightforward PCR amplifications, Sanger sequencing and blast analysis, the proposed strategy was demonstrated to be convenient for implementation in enforcement laboratories.

VL - 305 M3 - 10.1016/j.foodchem.2019.125431 ER - TY - JOUR T1 - Use of Whole Genome Sequencing Data for a First in Silico Specificity Evaluation of the RT-qPCR Assays Used for SARS-CoV-2 Detection. JF - Int J Mol Sci Y1 - 2020 A1 - Mathieu Gand A1 - Kevin Vanneste A1 - Isabelle Thomas A1 - Steven Van Gucht A1 - Arnaud Capron A1 - Philippe Herman A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - Betacoronavirus KW - Coronavirus Infections KW - Databases, Genetic KW - Genome, Viral KW - Humans KW - Open Reading Frames KW - Pandemics KW - Pneumonia, Viral KW - Polymorphism, Single Nucleotide KW - Real-Time Polymerase Chain Reaction KW - RNA Replicase KW - RNA, Viral KW - Sensitivity and Specificity KW - whole genome sequencing AB -

The current COronaVIrus Disease 2019 (COVID-19) pandemic started in December 2019. COVID-19 cases are confirmed by the detection of SARS-CoV-2 RNA in biological samples by RT-qPCR. However, limited numbers of SARS-CoV-2 genomes were available when the first RT-qPCR methods were developed in January 2020 for initial in silico specificity evaluation and to verify whether the targeted loci are highly conserved. Now that more whole genome data have become available, we used the bioinformatics tool SCREENED and a total of 4755 publicly available SARS-CoV-2 genomes, downloaded at two different time points, to evaluate the specificity of 12 RT-qPCR tests (consisting of a total of 30 primers and probe sets) used for SARS-CoV-2 detection and the impact of the virus' genetic evolution on four of them. The exclusivity of these methods was also assessed using the human reference genome and 2624 closely related other respiratory viral genomes. The specificity of the assays was generally good and stable over time. An exception is the first method developed by the China Center for Disease Control and prevention (CDC), which exhibits three primer mismatches present in 358 SARS-CoV-2 genomes sequenced mainly in Europe from February 2020 onwards. The best results were obtained for the assay of Chan et al. (2020) targeting the gene coding for the spiking protein (S). This demonstrates that our user-friendly strategy can be used for a first in silico specificity evaluation of future RT-qPCR tests, as well as verifying that the former methods are still capable of detecting circulating SARS-CoV-2 variants.

VL - 21 CP - 15 M3 - 10.3390/ijms21155585 ER - TY - JOUR T1 - Whole-genome sequencing of serotype 4b isolated from ready-to-eat lentil salad in Algiers, Algeria. JF - New Microbes New Infect Y1 - 2020 A1 - R Drali A1 - A Deriet A1 - Bavo Verhaegen A1 - Sigrid C.J. De Keersmaecker A1 - N Botteldoorn A1 - Kevin Vanneste A1 - Nancy Roosens A1 - F Mouffok AB -

is a Gram-positive food-borne pathogen causing a serious threat for public health. Here we announce the whole genome sequence (3 011 693 bp) of serotype 4b, isolated from ready-to-eat lentil salad in Algiers and belonging to sequence type 2, lineage I and clonal complex 2.

VL - 33 M3 - 10.1016/j.nmni.2019.100628 ER - TY - JOUR T1 - Detailed Evaluation of Data Analysis Tools for Subtyping of Bacterial Isolates Based on Whole Genome Sequencing: as a Proof of Concept. JF - Front Microbiol Y1 - 2019 A1 - Assia Saltykova A1 - Wesley Mattheus A1 - Sophie Bertrand A1 - Nancy Roosens A1 - Marchal, Kathleen A1 - Sigrid C.J. De Keersmaecker AB -

Whole genome sequencing is increasingly recognized as the most informative approach for characterization of bacterial isolates. Success of the routine use of this technology in public health laboratories depends on the availability of well-characterized and verified data analysis methods. However, multiple subtyping workflows are now often being used for a single organism, and differences between them are not always well described. Moreover, methodologies for comparison of subtyping workflows, and assessment of their performance are only beginning to emerge. Current work focuses on the detailed comparison of WGS-based subtyping workflows and evaluation of their suitability for the organism and the research context in question. We evaluated the performance of pipelines used for subtyping of , including the currently widely applied cgMLST approach and different SNP-based methods. In addition, the impact of the use of different tools for detection and filtering of recombinant regions and of different reference genomes were tested. Our benchmarking analysis included both assessment of technical performance of the pipelines and functional comparison of the generated genetic distance matrices and phylogenetic trees. It was carried out using replicate sequencing datasets of high- and low-coverage, consisting mainly of isolates belonging to the clonal complex 269. We demonstrated that cgMLST and some of the SNP-based subtyping workflows showed very good performance characteristics and highly similar genetic distance matrices and phylogenetic trees with isolates belonging to the same clonal complex. However, only two of the tested workflows demonstrated reproducible results for a group of more closely related isolates. Additionally, results of the SNP-based subtyping workflows were to some level dependent on the reference genome used. Interestingly, the use of recombination-filtering software generally reduced the similarity between the gene-by-gene and SNP-based methodologies for subtyping of . Our study, where was taken as an example, clearly highlights the need for more benchmarking comparative studies to eventually contribute to a justified use of a specific WGS data analysis workflow within an international public health laboratory context.

VL - 10 M3 - 10.3389/fmicb.2019.02897 ER - TY - JOUR T1 - Development of a real-time PCR method for the genoserotyping of Salmonella Paratyphi B variant Java. JF - Appl Microbiol Biotechnol Y1 - 2019 A1 - Mathieu Gand A1 - Wesley Mattheus A1 - Assia Saltykova A1 - Nancy Roosens A1 - Katelijne Dierick A1 - Marchal, Kathleen A1 - Sigrid C.J. De Keersmaecker A1 - Sophie Bertrand AB -

Discriminating between D-tartrate fermenting and non-fermenting strains of Salmonella enterica subsp. enterica serotype Paratyphi B is of major importance as these two variants have different pathogenic profiles. While D-tartrate non-fermenting S. Paratyphi B isolates are the causative agent of typhoid-like fever, D-tartrate fermenting isolates (also called variant Java) of the same serotype trigger the less dangerous gastroenteritis. The determination of S. Paratyphi B variants requires a time-consuming process and complex biochemical tests. Therefore, a quadruplex real-time PCR method, based on the allelic discrimination of molecular markers selected from the scientific literature and from whole genome sequencing data produced in-house, was developed in this study, to be applied to Salmonella isolates. This method was validated with the analysis of 178 S. Paratyphi B (D-tartrate fermenting and non-fermenting) and other serotypes reaching an accuracy, compared with the classical methods, of 98% for serotyping by slide agglutination and 100% for replacement of the biochemical test. The developed real-time PCR permits to save time and to obtain an accurate identification of a S. Paratyphi B serotype and its D-tartrate fermenting profile, which is needed in routine laboratories for fast and efficient diagnostics.

VL - 103 CP - 12 M3 - 10.1007/s00253-019-09854-4 ER - TY - JOUR T1 - Exploiting the Advantages of Molecular Tools for the Monitoring of Fungal Indoor Air Contamination: First Detection of in Indoor Air of Air-Conditioned Offices. JF - Microorganisms Y1 - 2019 A1 - Libert, Xavier A1 - Camille Chasseur A1 - Ann Packeu A1 - Bureau, Fabrice A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker AB -

Today, indoor air pollution is considered a public health issue. Among the impacting pollutants, indoor airborne fungi are increasingly highlighted. Most of the monitoring protocols are culture-based, but these are unable to detect the uncultivable and/or dead fraction or species suppressed by fast-growing fungi, even though this fraction could impact health. Among the contaminants suspected to be part of this fraction, is an interesting case study. Known to be pathogenic, this black yeast grows in humid environments such as air-conditioning systems, where it has been previously detected using classical culture-based methods. However, until now, this fungus was never detected in indoor air in contact with these air-conditioning systems. This study shows the first detection of in indoor air collected from offices in contact with contaminated air-conditioning reservoirs. While its presence in indoor air could not be demonstrated with culture-based methods, it was found by real-time PCR and massive parallel sequencing. The latter also allowed obtaining a broader view on the fungal diversity in the tested samples. Similar approaches were applied on water samples collected from the conditioning reservoirs to trace the source of contamination. The comparison of results obtained with both methods confirmed that the molecular tools could improve indoor air monitoring, especially of dead and/or uncultivable contaminants or when competition between species could occur.

VL - 7 CP - 12 M3 - 10.3390/microorganisms7120674 ER - TY - JOUR T1 - MinION sequencing technology to characterize unauthorized GM petunia plants circulating on the European Union market JF - Scientific reports Y1 - 2019 A1 - Marie-Alice Fraiture A1 - Gabriella Ujhelyi A1 - Jaroslava Ovesna A1 - D. Van Geel A1 - Sigrid C.J. De Keersmaecker A1 - Assia Saltykova A1 - N. Papazova A1 - Nancy Roosens KW - amplicon sequencing KW - detection KW - DNA walking KW - GM petunia KW - Oxford Nanopore Technologies KW - Real-time PCR AB -

In order to characterize unauthorized genetically modified petunia, an integrated strategy has been applied here on several suspected petunia samples from the European market. More precisely, DNA fragments of interest were produced by DNA walking anchored on key targets, earlier detected by real-time PCR screening analysis, to be subsequently sequenced using the MinION platform from Oxford Nanopore Technologies. This way, the presence of genetically modified petunia was demonstrated via the characterization of their transgene flanking regions as well as unnatural associations of elements from their transgenic cassette.

VL - 9 CP - 7141 M3 - https://doi.org/10.1038/s41598-019-43463-5 ER - TY - JOUR T1 - A new multiplex RT-qPCR method for the simultaneous detection and discrimination of Zika and chikungunya viruses. JF - Int J Infect Dis Y1 - 2019 A1 - Sylvia Broeders A1 - Linda Garlant A1 - Marie-Alice Fraiture A1 - Els Vandermassen A1 - Vanessa Suin A1 - Jessica Vanhomwegen A1 - Myrielle Dupont-Rouzeyrol A1 - Dominique Rousset A1 - Steven Van Gucht A1 - Nancy Roosens KW - chikungunya KW - VIRUS KW - Zika AB -

OBJECTIVE: The re-emergence and spreading of tropical viruses to new areas raised worldwide a wave of concern. To treat patients in an early stage and prevent diffusion of the outbreak, an early diagnosis, and thus fast and adequate detection, is needed. To this aim, a multiplex reverse transcription real-time polymerase chain reaction TaqMan® method was designed to detect universally Zika and chikungunya viruses.

DESIGN: Two methods, targeting different genome segments, were selected from literature for each virus, adapted for high genome coverage and joined into a four-plex assay which was thoroughly in-house validated. The SCREENED tool was used to evaluate the sequence coverage of the method.

RESULTS: The full validation approach showed that the new four-plex method allows specific and sensitive identification and discrimination of Zika and chikungunya in routine samples. The combination of two targets per virus allowing almost 100% coverage of about 500 genomes was shown for the first time.

CONCLUSION: PCR being a reliable user-friendly technique, applicable in remote areas, such multiplex methods enable early and efficient diagnosis leading to rapid treatment and effective confinement in outbreak cases as well as being an aid for surveillance and the full validation permits easy method-transfer allowing worldwide harmonization.

M3 - 10.1016/j.ijid.2019.12.028 ER - TY - JOUR T1 - Shifting national surveillance of Shigella infections toward geno-serotyping by the development of a tailored Luminex assay and NGS workflow. JF - Microbiologyopen Y1 - 2019 A1 - Eleonora Ventola A1 - Bert Bogaerts A1 - Sigrid C.J. De Keersmaecker A1 - Kevin Vanneste A1 - Nancy Roosens A1 - Wesley Mattheus A1 - Pieter-Jan Ceyssens KW - Luminex KW - multiplex KW - Public Health Surveillance KW - sequencing KW - Shigella AB -

The phylogenetically closely related Shigella species and enteroinvasive Escherichia coli (EIEC) are responsible for millions of episodes of bacterial dysenteriae worldwide. Given its distinct epidemiology and public health relevance, only Shigellae are subject to mandatory reporting and follow-up by public health authorities. However, many clinical laboratories struggle to differentiate non-EIEC, EIEC, and Shigella in their current workflows, leading to inaccuracies in surveillance and rising numbers of misidentified E. coli samples at the National Reference Centre (NRC). In this paper, we describe two novel tools to enhance Shigella surveillance. First, we developed a low-cost Luminex-based multiplex assay combining five genetic markers for species identification with 11 markers for serotype prediction for S. sonnei and S. flexneri isolates. Using a test panel of 254 clinical samples, this assay has a sensitivity of 100% in differentiation of EIEC/Shigella pathotype from non-EIEC strains, and 68.7% success rate in distinction of Shigella and EIEC. A novel, and particularly successful marker was a Shigella-specific deletion in the spermidine acetyltransferase gene speG, reflecting its metabolic decay. For Shigella serotype prediction, the multiplex assay scored a sensitivity and specificity of 96.6% and 98.4%, respectively. All discrepancies were analyzed with whole-genome sequencing and shown to be related to causative mutations (stop codons, indels, and promoter mutations) in glycosyltransferase genes. This observation spurred the development of an in silico workflow which extracts the Shigella serotype from Next-Generation Sequencing (NGS) data, taking into account gene functionality. Both tools will be implemented in the workflow of the NRC, and will play a major role in the shift from phenotypic to genotyping-based surveillance of shigellosis in Belgium.

M3 - 10.1002/mbo3.807 ER - TY - RPRT T1 - SPECENZYM : A project to study the purity of food enzymes Y1 - 2019 A1 - Marie Deckers A1 - Kevin Vanneste A1 - Sarah Denayer A1 - Sigrid C.J. De Keersmaecker A1 - M Heyndrickx A1 - M De Loose A1 - G Berben A1 - F Debode A1 - N. Papazova A1 - P Fox A1 - Laure Joly A1 - Ann Ruttens A1 - Karlien Cheyns A1 - Bart Huybrechts A1 - Emmanuel Tangni A1 - Didier Breyer A1 - D Deforce A1 - Marie-Alice Fraiture A1 - Nancy Roosens ER - TY - JOUR T1 - Status and potential of bacterial genomics for public health practice: a scoping review JF - Implementation Science Y1 - 2019 A1 - Nina Van Goethem A1 - Tine Descamps A1 - Brecht Devleesschauwer A1 - Nancy Roosens A1 - Nele Boon A1 - Herman Van Oyen A1 - Robert, Annie AB -

Background

Next-generation sequencing (NGS) is increasingly being translated into routine public health practice, affecting the surveillance and control of many pathogens. The purpose of this scoping review is to identify and characterize the recent literature concerning the application of bacterial pathogen genomics for public health practice and to assess the added value, challenges, and needs related to its implementation from an epidemiologist’s perspective.

Methods

In this scoping review, a systematic PubMed search with forward and backward snowballing was performed to identify manuscripts in English published between January 2015 and September 2018. Included studies had to describe the application of NGS on bacterial isolates within a public health setting. The studied pathogen, year of publication, country, number of isolates, sampling fraction, setting, public health application, study aim, level of implementation, time orientation of the NGS analyses, and key findings were extracted from each study. Due to a large heterogeneity of settings, applications, pathogens, and study measurements, a descriptive narrative synthesis of the eligible studies was performed.

Results

Out of the 275 included articles, 164 were outbreak investigations, 70 focused on strategy-oriented surveillance, and 41 on control-oriented surveillance. Main applications included the use of whole-genome sequencing (WGS) data for (1) source tracing, (2) early outbreak detection, (3) unraveling transmission dynamics, (4) monitoring drug resistance, (5) detecting cross-border transmission events, (6) identifying the emergence of strains with enhanced virulence or zoonotic potential, and (7) assessing the impact of prevention and control programs. The superior resolution over conventional typing methods to infer transmission routes was reported as an added value, as well as the ability to simultaneously characterize the resistome and virulome of the studied pathogen. However, the full potential of pathogen genomics can only be reached through its integration with high-quality contextual data.

Conclusions

For several pathogens, it is time for a shift from proof-of-concept studies to routine use of WGS during outbreak investigations and surveillance activities. However, some implementation challenges from the epidemiologist’s perspective remain, such as data integration, quality of contextual data, sampling strategies, and meaningful interpretations. Interdisciplinary, inter-sectoral, and international collaborations are key for an appropriate genomics-informed surveillance.

VL - 14 ER - TY - JOUR T1 - Status and potential of bacterial genomics for public health practice: a scoping review JF - Implementation Science Y1 - 2019 A1 - Nina Van Goethem A1 - Tine Descamps A1 - Brecht Devleesschauwer A1 - Nancy Roosens A1 - Nele AM Boon A1 - Herman Van Oyen A1 - Annie Robert VL - 14 CP - 1 M3 - 10.1186/s13012-019-0930-2 ER - TY - JOUR T1 - Targeting the 16S rRNA gene for bacterial identification in complex mixed samples: comparative evaluation of second (Illumina) and third (Oxford Nanopore Technologies) generation sequencing technologies. JF - Int J Mol Sci Y1 - 2019 A1 - Raf Winand A1 - Bert Bogaerts A1 - Stefan Hoffman A1 - Lefèvre, Loic A1 - Delvoye, Maud A1 - Julien Van Braekel A1 - Fu, Qiang A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker A1 - Kevin Vanneste KW - bacteria KW - DNA, Bacterial KW - High-Throughput Nucleotide Sequencing KW - Nanopores KW - RNA, Ribosomal, 16S AB -

Rapid, accurate bacterial identification in biological samples is an important task for microbiology laboratories, for which 16S~rRNA gene Sanger sequencing of cultured isolates is frequently used. In contrast, next-generation sequencing does not require intermediate culturing steps and can be directly applied on communities, but its performance has not been extensively evaluated. We present a comparative evaluation of second (Illumina) and third (Oxford Nanopore Technologies (ONT)) generation sequencing technologies for 16S targeted genomics using a well-characterized reference sample. Different 16S gene regions were amplified and sequenced using the Illumina MiSeq, and analyzed with Mothur. Correct classification was variable, depending on the region amplified. Using a majority vote over all regions, most false positives could be eliminated at the genus level but not the species level. Alternatively, the entire 16S gene was amplified and sequenced using the ONT MinION, and analyzed with Mothur, EPI2ME, and GraphMap. Although >99\% of reads were correctly classified at the genus level, up to $\approx$40\% were misclassified at the species level. Both~technologies, therefore, allow reliable identification of bacterial genera, but can potentially misguide identification of bacterial species, and constitute viable alternatives to Sanger sequencing for rapid analysis of mixed samples without requiring any culturing steps.

VL - 21 CP - 1 M3 - 10.3390/ijms21010298 ER - TY - JOUR T1 - Validation of a Bioinformatics Workflow for Routine Analysis of Whole-Genome Sequencing Data and Related Challenges for Pathogen Typing in a European National Reference Center: as a Proof-of-Concept. JF - Front Microbiol Y1 - 2019 A1 - Bert Bogaerts A1 - Raf Winand A1 - Fu, Qiang A1 - Julien Van Braekel A1 - Pieter-Jan Ceyssens A1 - Wesley Mattheus A1 - Sophie Bertrand A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Kevin Vanneste KW - national reference center KW - Neisseria meningitidis KW - public health KW - VALIDATION KW - whole-genome sequencing AB -

Despite being a well-established research method, the use of whole-genome sequencing (WGS) for routine molecular typing and pathogen characterization remains a substantial challenge due to the required bioinformatics resources and/or expertise. Moreover, many national reference laboratories and centers, as well as other laboratories working under a quality system, require extensive validation to demonstrate that employed methods are "fit-for-purpose" and provide high-quality results. A harmonized framework with guidelines for the validation of WGS workflows does currently, however, not exist yet, despite several recent case studies highlighting the urgent need thereof. We present a validation strategy focusing specifically on the exhaustive characterization of the bioinformatics analysis of a WGS workflow designed to replace conventionally employed molecular typing methods for microbial isolates in a representative small-scale laboratory, using the pathogen as a proof-of-concept. We adapted several classically employed performance metrics specifically toward three different bioinformatics assays: resistance gene characterization (based on the ARG-ANNOT, ResFinder, CARD, and NDARO databases), several commonly employed typing schemas (including, among others, core genome multilocus sequence typing), and serogroup determination. We analyzed a core validation dataset of 67 well-characterized samples typed by means of classical genotypic and/or phenotypic methods that were sequenced in-house, allowing to evaluate repeatability, reproducibility, accuracy, precision, sensitivity, and specificity of the different bioinformatics assays. We also analyzed an extended validation dataset composed of publicly available WGS data for 64 samples by comparing results of the different bioinformatics assays against results obtained from commonly used bioinformatics tools. We demonstrate high performance, with values for all performance metrics >87%, >97%, and >90% for the resistance gene characterization, sequence typing, and serogroup determination assays, respectively, for both validation datasets. Our WGS workflow has been made publicly available as a "push-button" pipeline for Illumina data at https://galaxy.sciensano.be to showcase its implementation for non-profit and/or academic usage. Our validation strategy can be adapted to other WGS workflows for other pathogens of interest and demonstrates the added value and feasibility of employing WGS with the aim of being integrated into routine use in an applied public health setting.

VL - 10 M3 - 10.3389/fmicb.2019.00362 ER - TY - JOUR T1 - Whole-Genome Sequencing of Multidrug-Resistant Escherichia coli Strains Harboring the Gene, Isolated from Seawater of the Algiers Coast in Algeria. JF - Microbiol Resour Announc Y1 - 2019 A1 - M Berrazeg A1 - A Deriet A1 - Sigrid C.J. De Keersmaecker A1 - Bavo Verhaegen A1 - Kevin Vanneste A1 - N Botteldoorn A1 - Nancy Roosens A1 - F Mouffok A1 - R Drali AB -

Colistin resistance has emerged worldwide and is threatening the treatment efficacy of multiresistant strains in humans and animals. Here, we communicate the whole-genome sequencing (WGS) of two colistin-resistant strains, M49 and M78, with genomes sizes of 4,947,168 and 5,178,716 bp, respectively, isolated from seawaters of the Algiers coast.

VL - 8 CP - 34 M3 - 10.1128/MRA.00638-19 ER - TY - JOUR T1 - Whole-Genome Sequencing of Six Strains of Salmonella enterica Isolated from Imported Meat in Algeria. JF - Microbiol Resour Announc Y1 - 2019 A1 - A Deriet A1 - M Berrazeg A1 - Sigrid C.J. De Keersmaecker A1 - N Botteldoorn A1 - Kevin Vanneste A1 - Bavo Verhaegen A1 - Nancy Roosens A1 - F Mouffok A1 - R Drali AB -

Nontyphoidal (NTS) is one of the main causes of foodborne disease worldwide. In this report, we announce the first whole-genome sequencing of six strains of isolated from imported meat in Algeria. The genome sizes ranged from 4,601,209 to 4,958,962 bp. Antimicrobial resistance (AMR) genes, plasmids, and virulence factors were detected.

VL - 8 CP - 35 M3 - 10.1128/MRA.00615-19 ER - TY - JOUR T1 - Application of whole genome data for in silico evaluation of primers and probes routinely employed for the detection of viral species by RT-qPCR using dengue virus as a case study. JF - BMC Bioinformatics Y1 - 2018 A1 - Kevin Vanneste A1 - Linda Garlant A1 - Sylvia Broeders A1 - Steven Van Gucht A1 - Nancy Roosens AB -

BACKGROUND: Viral infection by dengue virus is a major public health problem in tropical countries. Early diagnosis and detection are increasingly based on quantitative reverse transcriptase real-time polymerase chain reaction (RT-qPCR) directed against genomic regions conserved between different isolates. Genetic variation can however result in mismatches of primers and probes with their targeted nucleic acid regions. Whole genome sequencing allows to characterize and track such changes, which in turn enables to evaluate, optimize, and (re-)design novel and existing RT-qPCR methods. The immense amount of available sequence data renders this however a labour-intensive and complex task.

RESULTS: We present a bioinformatics approach that enables in silico evaluation of primers and probes intended for routinely employed RT-qPCR methods. This approach is based on analysing large amounts of publically available whole genome data, by first employing BLASTN to mine the genomic regions targeted by the RT-qPCR method(s), and afterwards using BLASTN-SHORT to evaluate whether primers and probes will anneal based on a set of simple in silico criteria. Using dengue virus as a case study, we evaluated 18 published RT-qPCR methods using more than 3000 publically available genomes in the NCBI Virus Variation Resource, and provide a systematic overview of method performance based on in silico sensitivity and specificity.

CONCLUSIONS: We provide a comprehensive overview of dengue virus RT-qPCR method performance that will aid appropriate method selection allowing to take specific measures that aim to contain and prevent viral spread in afflicted regions. Notably, we find that primer-template mismatches at their 3' end may represent a general issue for dengue virus RT-qPCR detection methods that merits more attention in their development process. Our approach is also available as a public tool, and demonstrates how utilizing genomic data can provide meaningful insights in an applied public health setting such as the detection of viral species in human diagnostics.

VL - 19 CP - 1 M3 - 10.1186/s12859-018-2313-0 ER - TY - JOUR T1 - Comparison of SNP-based subtyping workflows for bacterial isolates using WGS data, applied to Salmonella enterica serotype Typhimurium and serotype 1,4,[5],12:i:. JF - PLoS One Y1 - 2018 A1 - Assia Saltykova A1 - Wuyts, Véronique A1 - Wesley Mattheus A1 - Sophie Bertrand A1 - Nancy Roosens A1 - Marchal, Kathleen A1 - Sigrid C.J. De Keersmaecker KW - bacterial phylogeny; food-borne; genes; NGS; WGS; computational biology/bioinformatics; disease outbreaks; outbreak; performance evaluation; pipeline; Salmonella; Salmonella enterica; Salmonella typhimurium; SNP; subtyping; whole genome sequencing AB -

Whole genome sequencing represents a promising new technology for subtyping of bacterial pathogens. Besides the technological advances which have pushed the approach forward, the last years have been marked by considerable evolution of the whole genome sequencing data analysis methods. Prior to application of the technology as a routine epidemiological typing tool, however, reliable and efficient data analysis strategies need to be identified among the wide variety of the emerged methodologies. In this work, we have compared three existing SNP-based subtyping workflows using a benchmark dataset of 32 Salmonella enterica subsp. enterica serovar Typhimurium and serovar 1,4,[5],12:i:- isolates including five isolates from a confirmed outbreak and three isolates obtained from the same patient at different time points. The analysis was carried out using the original (high-coverage) and a down-sampled (low-coverage) datasets and two different reference genomes. All three tested workflows, namely CSI Phylogeny-based workflow, CFSAN-based workflow and PHEnix-based workflow, were able to correctly group the confirmed outbreak isolates and isolates from the same patient with all combinations of reference genomes and datasets. However, the workflows differed strongly with respect to the SNP distances between isolates and sensitivity towards sequencing coverage, which could be linked to the specific data analysis strategies used therein. To demonstrate the effect of particular data analysis steps, several modifications of the existing workflows were also tested. This allowed us to propose data analysis schemes most suitable for routine SNP-based subtyping applied to S. Typhimurium and S. 1,4,[5],12:i:-. Results presented in this study illustrate the importance of using correct data analysis strategies and to define benchmark and fine-tune parameters applied within routine data analysis pipelines to obtain optimal results.

VL - 13 CP - 2 M3 - 10.1371/journal.pone.0192504 ER - TY - JOUR T1 - Detection and discrimination of five E. coli pathotypes using a combinatory SYBR® Green qPCR screening system JF - Applied Microbiology and Biotechnology Y1 - 2018 A1 - Barbau-Piednoir, Elodie A1 - Sarah Denayer A1 - N Botteldoorn A1 - Katelijne Dierick A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens KW - Detection; Pathogenic E. coli; Real-time PCR; STEC; SYBR® Green; Validation AB -

A detection and discrimination system for five Escherichia coli pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic E. coli (CoSYPS Path E. coli). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of E. coli: shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) E. coli. The SYBR® Green qPCR assays target the uidA, ipaH, eae, aggR, aaiC, stx1, and stx2 genes. uidA controls for E. coli presence and all the other genes are specific targets of E. coli pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs' design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path E. coli system was subsequently evaluated on four food matrices artificially contaminated with pathogenic E. coli. It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.

VL - 102 CP - 7 M3 - 10.1007/s00253-018-8820-0 ER - TY - JOUR T1 - Development and validation of an integrated DNA walking strategy to detect GMO expressing cry genes JF - BMC Biotechnology Y1 - 2018 A1 - Marie-Alice Fraiture A1 - Julie Vandamme A1 - Philippe Herman A1 - Nancy Roosens KW - Characterization KW - detection KW - DNA walking KW - GMO KW - identification KW - qPCR AB -

Background

Recently, an integrated DNA walking strategy has been proposed to prove the presence of GMO via the characterisation of sequences of interest, including their transgene flanking regions and the unnatural associations of elements in their transgenic cassettes. To this end, the p35S, tNOS and t35S pCAMBIA elements have been selected as key targets, allowing the coverage of most of GMO, EU authorized or not. In the present study, a bidirectional DNA walking method anchored on the CryAb/c genes is proposed with the aim to cover additional GMO and additional sequences of interest.

Results

The performance of the proposed bidirectional DNA walking method anchored on the CryAb/c genes has been evaluated in a first time for its feasibility using several GM events possessing these CryAb/c genes. Afterwards, its sensitivity has been investigated through low concentrations of targets (as low as 20 HGE). In addition, to illustrate its applicability, the entire workflow has been tested on a sample mimicking food/feed matrices analysed in GMO routine analysis.

Conclusion

Given the successful assessment of its performance, the present bidirectional DNA walking method anchored on the CryAb/c genes can easily be implemented in GMO routine analysis by the enforcement laboratories and allows completing the entire DNA walking strategy in targeting an additional transgenic element frequently found in GMO.

VL - 18 CP - 1 M3 - 10.1186/s12896-018-0446-x ER - TY - Generic T1 - Development of a bioinformatics pipeline for the routine analysis of Influenza whole genome sequencing data Y1 - 2018 A1 - Qiang Fu A1 - Raf Winand A1 - Julien Van Braekel A1 - Cyril Barbezange A1 - Veronik Hutse A1 - Isabelle Thomas A1 - Steven Van Gucht A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - Kevin Vanneste JF - European Conference on Computational Biology 2018 conference (http://eccb18.org/). 8 – 12 September, 2018, Athens, Greece ER - TY - JOUR T1 - The genetic structure of the Belgian population JF - Human Genomics Y1 - 2018 A1 - Jimmy Van den Eynden A1 - Tine Descamps A1 - Els Delporte A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker A1 - Vanessa De Wit A1 - Joris Robert Vermeesch A1 - Goetghebeur, Els A1 - Jean Tafforeau A1 - Stefaan Demarest A1 - Marc Van den Bulcke A1 - Herman Van Oyen KW - genetic variability KW - population genomics KW - public health genomics VL - 12 CP - 1 M3 - 10.1186/s40246-018-0136-8 ER - TY - JOUR T1 - High-resolution melting PCR analysis for rapid genotyping of Burkholderia mallei. JF - Infect Genet Evol Y1 - 2018 A1 - G Girault A1 - P Wattiau A1 - M Saqib A1 - B Martin A1 - F Vorimore A1 - H Singha A1 - M Engelsma A1 - H J Roest A1 - S Spicic A1 - R Grunow A1 - N Vicari A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens A1 - M Fabbi A1 - B N Tripathi A1 - Zientara, S A1 - N Madani A1 - K Laroucau KW - Burkholderia mallei KW - DNA, Bacterial KW - Genotype KW - Phylogeny KW - polymerase chain reaction KW - Polymorphism, Single Nucleotide AB -

Burkholderia (B.) mallei is the causative agent of glanders. A previous work conducted on single-nucleotide polymorphisms (SNP) extracted from the whole genome sequences of 45 B. mallei isolates identified 3 lineages for this species. In this study, we designed a high-resolution melting (HRM) method for the screening of 15 phylogenetically informative SNPs within the genome of B. mallei that subtype the species into 3 lineages and 12 branches/sub-branches/groups. The present results demonstrate that SNP-based genotyping represent an interesting approach for the molecular epidemiology analysis of B. mallei.

VL - 63 M3 - 10.1016/j.meegid.2018.05.004 ER - TY - Generic T1 - Monitoring of influenza: Whole-Genome Sequencing to provide insights in the disease severity Y1 - 2018 A1 - Laura Van Poelvoorde A1 - Sigrid C.J. De Keersmaecker A1 - Kevin Vanneste A1 - Steven Van Gucht A1 - Isabelle Thomas A1 - Xavier Saelens A1 - Cyril Barbezange A1 - Nancy Roosens JF - The 6th International Influenza Meeting, 2-4 September 2018 Münster, Germany ER - TY - Generic T1 - A multiplex oligonucleotide ligation-PCR method using the Luminex technology for the geno-serotyping of the most common Salmonella in Belgium Y1 - 2018 A1 - Mathieu Gand A1 - Wesley Mattheus A1 - Assia Saltykova A1 - Nancy Roosens A1 - Katelijne Dierick A1 - Marchal, Kathleen A1 - Sigrid C.J. De Keersmaecker A1 - Sophie Bertrand KW - Luminex KW - method KW - multiplex KW - Salmonella KW - Serotyping JF - ECCMID PB - ESCMID CY - Madrid, Spain ER - TY - JOUR T1 - Nanopore sequencing technology: a new route for the fast detection of unauthorized GMO. JF - Sci Rep Y1 - 2018 A1 - Marie-Alice Fraiture A1 - Assia Saltykova A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens ED - Hoffmann,S. ED - Raf Winand ED - Deforce, Dieter ED - Kevin Vanneste KW - DNA walking KW - food and feed safety KW - GMO KW - GMO detection KW - method KW - method development KW - nanopore sequencing KW - Unauthorised GMO AB -

In order to strengthen the current genetically modified organism (GMO) detection system for unauthorized GMO, we have recently developed a new workflow based on DNA walking to amplify unknown sequences surrounding a known DNA region. This DNA walking is performed on transgenic elements, commonly found in GMO, that were earlier detected by real-time PCR (qPCR) screening. Previously, we have demonstrated the ability of this approach to detect unauthorized GMO via the identification of unique transgene flanking regions and the unnatural associations of elements from the transgenic cassette. In the present study, we investigate the feasibility to integrate the described workflow with the MinION Next-Generation-Sequencing (NGS). The MinION sequencing platform can provide long read-lengths and deal with heterogenic DNA libraries, allowing for rapid and efficient delivery of sequences of interest. In addition, the ability of this NGS platform to characterize unauthorized and unknown GMO without any a priori knowledge has been assessed.

VL - 8 CP - 1 M3 - 10.1038/s41598-018-26259-x ER - TY - JOUR T1 - A novel genotoxin-specific qPCR array based on the metabolically competent human HepaRG™ cell line as a rapid and reliable tool for improved in vitro hazard assessment JF - Archives of Toxicology Y1 - 2018 A1 - Ates, Gamze A1 - Birgit Mertens A1 - Heymans, Anja A1 - Luc Verschaeve A1 - Milushev, Dimiter A1 - Vanparys, Philippe A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker A1 - Rogiers, Vera A1 - Tatyana Y. Doktorova KW - genotoxicity KW - HepaRG KW - In vitro screening KW - mutagenicity KW - qPCR array KW - Toxicogenomics AB -

Although the value of the regulatory accepted batteries for in vitro genotoxicity testing is recognized, they result in a high

number of false positives. This has a major impact on society and industries developing novel compounds for pharmaceutical,

chemical, and consumer products, as afflicted compounds have to be (prematurely) abandoned or further tested on

animals. Using the metabolically competent human HepaRG

™ cell line and toxicogenomics approaches, we have developed

an upgraded, innovative, and proprietary gene classifier. This gene classifier is based on transcriptomic changes induced by

12 genotoxic and 12 non-genotoxic reference compounds tested at sub-cytotoxic concentrations, i.e., IC10 concentrations

as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The resulting gene classifier

was translated into an easy-to-handle qPCR array that, as shown by pathway analysis, covers several different cellular

processes related to genotoxicity. To further assess the predictivity of the tool, a set of 5 known positive and 5 known negative

test compounds for genotoxicity was evaluated. In addition, 2 compounds with debatable genotoxicity data were tested

to explore how the qPCR array would classify these. With an accuracy of 100%, when equivocal results were considered

positive, the results showed that combining HepaRG

™ cells with a genotoxin-specific qPCR array can improve (geno)toxicological

hazard assessment. In addition, the developed qPCR array was able to provide additional information on compounds

for which so far debatable genotoxicity data are available. The results indicate that the new in vitro tool can improve human

safety assessment of chemicals in general by basing predictions on mechanistic toxicogenomics information.

M3 - 10.1007/s00204-018-2172-5 ER - TY - JOUR T1 - Detection of Plasmid-Mediated Colistin Resistance, mcr-1 and mcr-2 Genes, in Salmonella spp. Isolated from Food at Retail in Belgium from 2012 to 2015. JF - Foodborne Pathog Dis Y1 - 2017 A1 - Cristina Garcia-Graells A1 - Sigrid C.J. De Keersmaecker A1 - Nguyen, Quang Trung A1 - Anthonissen,R.. A1 - Raf Aerts A1 - Nancy Roosens A1 - Katelijne Dierick A1 - N Botteldoorn KW - colistin resistance AB -

A collection of 105 colistin-resistant Salmonella isolates collected from 2012 to 2015 in the national surveillance program in Belgium was screened by PCR for the presence of genes mcr-1 and mcr-2. Of these, 1.90% (2/105) and 0.95% (1/105) tested positive for mcr-1 and mcr-2, respectively. The presence of the mcr-1 or mcr-2 determinant has been confirmed by whole genome sequencing and allowed the localization of these two genes on IncX4 type plasmids. We report here the presence of mcr-1 and the first mcr-2 gene in Salmonella ever isolated in the Belgian food chain. Although present at retail since 2012, the occurrence is low and sporadic.

M3 - 10.1089/fpd.2017.2329 ER - TY - JOUR T1 - Development and performance assessment of a luminex xMAP® direct hybridization assay for the detection and identification of indoor air fungal contamination. JF - PLoS One Y1 - 2017 A1 - Libert, Xavier A1 - Ann Packeu A1 - Bureau, Fabrice A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - Air Pollution, Indoor KW - DNA Probes KW - environment KW - Fungi KW - Limit of Detection KW - Nucleic Acid Hybridization AB -

Considered as a public health problem, indoor fungal contamination is generally monitored using classical protocols based on culturing. However, this culture dependency could influence the representativeness of the fungal population detected in an analyzed sample as this includes the dead and uncultivable fraction. Moreover, culture-based protocols are often time-consuming. In this context, molecular tools are a powerful alternative, especially those allowing multiplexing. In this study a Luminex xMAP® assay was developed for the simultaneous detection of 10 fungal species which are most frequently in indoor air and that may cause health problems. This xMAP® assay was found to be sensitive, i.e. its limit of detection is ranging between 0.05 and 0.01 ng of gDNA. The assay was subsequently tested with environmental air samples which were also analyzed with a classical protocol. All the species identified with the classical method were also detected with the xMAP® assay, however in a shorter time frame. These results demonstrate that the Luminex xMAP® fungal assay developed in this study could contribute to the improvement of public health and specifically to the indoor fungal contamination treatment.

VL - 12 CP - 3 U1 - http://www.ncbi.nlm.nih.gov/pubmed/28278219?dopt=Abstract M3 - 10.1371/journal.pone.0173390 ER - TY - Generic T1 - Development of a real-time PCR test for the geno-serotyping of Salmonella Paratyphi B and its variant Java Y1 - 2017 A1 - Mathieu Gand A1 - Wesley Mattheus A1 - Assia Saltykova A1 - Nancy Roosens A1 - Katelijne Dierick A1 - Marchal, Kathleen A1 - Sigrid C.J. De Keersmaecker A1 - Sophie Bertrand KW - Luminex KW - method KW - mutliplex KW - Salmonella KW - Serotyping JF - BSFM conference PB - BSFM CY - Brussels, Belgium ER - TY - JOUR T1 - Discrimination of three genetically close Aspergillus species by using high resolution melting analysis applied to indoor air as case study. JF - BMC Microbiol Y1 - 2017 A1 - Libert, Xavier A1 - Ann Packeu A1 - Bureau, Fabrice A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker AB -

BACKGROUND: Indoor air pollution caused by fungal contamination is suspected to have a public health impact. Monitoring of the composition of the indoor airborne fungal contaminants is therefore important. To avoid problems linked to culture-dependent protocols, molecular methods are increasingly being proposed as an alternative. Among these molecular methods, the polymerase chain reaction (PCR) and the real-time PCR are the most frequently used tools for indoor fungal detection. However, even if these tools have demonstrated their appropriate performance, some of them are not able to discriminate between species which are genetically close. A solution to this could be the use of a post-qPCR high resolution melting (HRM) analysis, which would allow the discrimination of these species based on the highly accurate determination of the difference in melting temperature of the obtained amplicon. In this study, we provide a proof-of-concept for this approach, using a dye adapted version of our previously developed qPCR SYBR®Green method to detect Aspergillus versicolor in indoor air, an important airborne fungus in terms of occurrence and cause of health problems. Despite the good performance observed for that qPCR method, no discrimination could previously be made between A. versicolor, Aspergillus creber and Aspergillus sydowii.

METHODS: In this study, we developed and evaluated an HRM assay for the discrimination between A. versicolor, Aspergillus creber and Aspergillus sydowii.

RESULTS: Using HRM analysis, the discrimination of the 3 Aspergillus species could be made. No false positive, nor false negatives were observed during the performance assessment including 20 strains of Aspergillus. The limit of detection was determined for each species i.e., 0.5 pg of gDNA for A. creber and A. sydowii, and 0.1 pg of gDNA for A. versicolor. The HRM analysis was also successfully tested on environmental samples.

CONCLUSION: We reported the development of HRM tools for the discrimination of A. versicolor, A. creber and A. sydowii. However, this study could be considered as a study case demonstrating that HRM based on existing qPCR assays, allows a more accurate identification of indoor air contaminants. This contributes to an improved insight in the diversity of indoor airborne fungi and hence, eventually in the causal link with health problems.

VL - 17 CP - 1 U1 - http://www.ncbi.nlm.nih.gov/pubmed/28376723?dopt=Abstract M3 - 10.1186/s12866-017-0996-4 ER - TY - Generic T1 - Evaluation of SNPs based on DNA extracted from urine as part of the development and use of a kit of non-invasive biomarkers to monitor respiratory health of young children Y1 - 2017 A1 - S. Nauwelaerts A1 - Koen De Cremer A1 - Bernard, Alfred A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - biomarkers; SNP KW - CHILDREN KW - non-invasive KW - respiratory health JF - qPCR dPCR & NGS 2017 Symposium PB - qPCR dPCR & NGS 2017 Symposium CY - Freising, Germany ER - TY - JOUR T1 - How can we better detect unauthorized GMO in the food and feed chain JF - Trends in Biotechnology Y1 - 2017 A1 - Marie-Alice Fraiture A1 - Philippe Herman A1 - De Loose, Marc A1 - Debode, Frédéric A1 - Nancy Roosens KW - detection KW - DNA walking KW - Monitoring KW - new workflow KW - next-generation-sequencing KW - qPCR KW - unauthorized GMO AB -

Current GMO detection systems have limited abilities to detect unauthorized genetically modified organisms (GMOs). Here, we propose a new workflow, based on next-generation sequencing (NGS) technology, to overcome this problem. In providing information about DNA sequences, this high-throughput workflow can distinguish authorized and unauthorized GMOs by strengthening the tools commonly used by enforcement laboratories with the help of NGS technology. In addition, thanks to its massive sequencing capacity, this workflow could be used to monitor GMOs present in the food and feed chain. In view of its potential implementation by enforcement laboratories, we discuss this innovative approach, its current limitations, and its sustainability of use over time.

VL - 35 CP - 6 M3 - https://doi.org/10.1016/j.tibtech.2017.03.002 ER - TY - Generic T1 - How to detect pathogens, leading to outbreaks, released accidentally or deliberately into the environment ? Y1 - 2017 A1 - Marie-Alice Fraiture A1 - Nancy Roosens A1 - Philippe Herman KW - How to detect pathogens KW - leading to outbreaks KW - released accidentally or deliberately into the environment ? PB - 7th Annual International Symposium on Biosecurity and Biosafety: future trends and solutions CY - Milan, Italy ER - TY - JOUR T1 - An integrated strategy combining DNA walking and NGS to detect GMO JF - Food Chemistry Y1 - 2017 A1 - Marie-Alice Fraiture A1 - Philippe Herman A1 - N. Papazova A1 - De Loose, Marc A1 - Deforce, Dieter A1 - Ruttink, Tom A1 - Nancy Roosens KW - detection KW - DNA walking KW - GMO KW - Next-generation sequencing KW - qPCR AB -

Recently, we developed a DNA walking system for the detection and characterization of a broad spectrum of GMOs in routine analysis of food/feed matrices. Here, we present a new version with improved throughput and sensitivity by coupling the DNA walking system to Pacific Bioscience® Next-generation sequencing technology. The performance of the new strategy was thoroughly assessed through several assays. First, we tested its detection and identification capability on grains with high or low GMO content. Second, the potential impacts of food processing were investigated using rice noodle samples. Finally, GMO mixtures and a real-life sample were analyzed to illustrate the applicability of the proposed strategy in routine GMO analysis. In all tested samples, the presence of multiple GMOs was unambiguously proven by the characterization of transgene flanking regions and the combinations of elements that are typical for transgene constructs.

VL - 232 M3 - https://doi.org/10.1016/j.foodchem.2017.03.067 ER - TY - JOUR T1 - Model-Based Classification for Digital PCR: Your Umbrella for Rain. JF - Anal Chem Y1 - 2017 A1 - Jacobs, Bart K M A1 - Goetghebeur, Els A1 - Vandesompele, Jo A1 - De Ganck, Ariane A1 - Nijs, Nele A1 - Beckers, Anneleen A1 - N. Papazova A1 - Nancy Roosens A1 - Clement, Lieven AB -

Standard data analysis pipelines for digital PCR estimate the concentration of a target nucleic acid by digitizing the end-point fluorescence of the parallel micro-PCR reactions, using an automated hard threshold. While it is known that misclassification has a major impact on the concentration estimate and substantially reduces accuracy, the uncertainty of this classification is typically ignored. We introduce a model-based clustering method to estimate the probability that the target is present (absent) in a partition conditional on its observed fluorescence and the distributional shape in no-template control samples. This methodology acknowledges the inherent uncertainty of the classification and provides a natural measure of precision, both at individual partition level and at the level of the global concentration. We illustrate our method on genetically modified organism, inhibition, dynamic range, and mutation detection experiments. We show that our method provides concentration estimates of similar accuracy or better than the current standard, along with a more realistic measure of precision. The individual partition probabilities and diagnostic density plots further allow for some quality control. An R implementation of our method, called Umbrella, is available, providing a more objective and automated data analysis procedure for absolute dPCR quantification.

VL - 89 CP - 8 U1 - http://www.ncbi.nlm.nih.gov/pubmed/28350455?dopt=Abstract M3 - 10.1021/acs.analchem.6b04208 ER - TY - JOUR T1 - Molecular Subtyping of Salmonella Typhimurium with Multiplex Oligonucleotide Ligation-PCR (MOL-PCR). JF - Methods Mol Biol Y1 - 2017 A1 - Wuyts, Véronique A1 - Wesley Mattheus A1 - Nancy Roosens A1 - Marchal, Kathleen A1 - Sophie Bertrand A1 - Sigrid C.J. De Keersmaecker AB -

A multiplex oligonucleotide ligation-PCR (MOL-PCR) assay is a valuable high-throughput technique for the detection of bacteria and viruses, for characterization of pathogens and for diagnosis of genetic diseases, as it allows one to combine different types of molecular markers in a high-throughput multiplex assay. A MOL-PCR assay starts with a multiplex oligonucleotide ligation reaction for detection of the molecular marker, followed by a singleplex PCR for signal amplification and analysis of the MOL-PCR products on a Luminex platform. This last step occurs through a liquid bead suspension array in which the MOL-PCR products are hybridized to MagPlex-TAG beads.In this chapter, we describe the complete procedure for a MOL-PCR assay for subtyping of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant S. 1,4[5],12:i:- from DNA isolation through heat lysis up to data interpretation through a Gödel Prime Product. The subtyping assay consists of 50 discriminative molecular markers and two internal positive control markers divided over three MOL-PCR assays.

VL - 1616 U1 - http://www.ncbi.nlm.nih.gov/pubmed/28600761?dopt=Abstract M3 - 10.1007/978-1-4939-7037-7_3 ER - TY - Generic T1 - Salmonella infections: identification techniques for successful investigations Y1 - 2017 A1 - Sophie Bertrand A1 - Wesley Mattheus A1 - Pieter-Jan Ceyssens A1 - Mathieu Gand A1 - Raymond Vanhoof A1 - N Botteldoorn A1 - Sarah Denayer A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - identification KW - method KW - Salmonella JF - Labinfo PB - AFSCA-FAVV CY - Belgium VL - 16 ER - TY - Generic T1 - The added value of whole genome sequencing for the development of a specific detection method for an EU-unauthorized genetically modified Bacillus subtilis overproducing riboflavin Y1 - 2016 A1 - Sigrid C.J. De Keersmaecker A1 - E. Barbau-Piednoir A1 - Delvoye,M. A1 - Gau,C. A1 - Philipp,P. A1 - Nancy Roosens KW - a KW - an KW - Bacillus KW - Bacillus subtilis KW - detection KW - detection method KW - Development KW - food KW - Food Safety KW - genetically KW - Genetically modified KW - Genome KW - GMM KW - health KW - Impact KW - management KW - meeting KW - method KW - NGS KW - ON KW - SAFETY KW - specific KW - whole genome KW - whole genome sequencing JF - Technical Meeting on the impact of Whole Genome Sequencing (WGS) on food safety management: within a one health framework PB - NA CY - NA CP - FAO/GMI U1 - 5281 U2 - 05/23-25/2016 ER - TY - BOOK T1 - Advances in the identification of genetically modified foods T2 - Advances in the Identification of Genetically Modified Foods Y1 - 2016 A1 - Marie-Alice Fraiture A1 - Sylvia Broeders A1 - Philippe Herman A1 - Taverniers, Isabel A1 - De Loose, Marc A1 - Deforce, Dieter A1 - Nancy Roosens KW - Advances in the Identification of Genetically Modified Foods JF - Advances in the Identification of Genetically Modified Foods PB - Elsevier Science CY - London, UK SN - 978-0-08-100220-9 CP - 20 ER - TY - RPRT T1 - Annual report of the GMOLAB concerning the validation dossiers for identification of genetically modified organisms (GMO) Y1 - 2016 A1 - N. Papazova A1 - Els Vandermassen A1 - Delvoye,M. A1 - D. Van Geel A1 - L. Lefèvre A1 - Van Duppen,J. A1 - Sylvia Broeders A1 - Nancy Roosens A1 - Sophie Carbonnelle A1 - Anne-Marie Vanherle KW - annual report KW - genetically KW - Genetically modified KW - Genetically modified organism KW - Genetically modified organisms KW - GMO KW - GMOlab KW - identification KW - report KW - VALIDATION PB - WIV-ISP CY - Brussels, Belgium SN - D/2016/2505/32 U1 - 5286 ER - TY - JOUR T1 - Biotech Rice: Current developments and future detection challenges in food and feed chain JF - Trends in Food Science & Technology Y1 - 2016 A1 - Marie-Alice Fraiture A1 - Nancy Roosens A1 - Taverniers, Isabel A1 - De Loose, Marc A1 - Deforce, Dieter A1 - Philippe Herman KW - Biotech crop KW - detection KW - Transgenic Rice AB -

Background

To improve agricultural practices and the food/feed security, plant breeding techniques were developed, including transgenesis commonly using Agrobacterium tumefaciens or biolistic technologies. To guarantee the traceability of GMO in food/feed chain and the consumer's freedom of choice, regulatory frameworks were established in many countries around the world, such as in Europe. Their implementations, including detection systems usually based on qPCR, are becoming complex and expensive regarding the number of analysis to perform. Moreover, the dispersion of publicly available information about developed GMO prevents to accurately estimate the efficiency of the standard detection system applied to unauthorized GMO.

Scope and approach

To illustrate this problem, the case of rice, one of the leading staple crops, was investigated. An overview of worldwide developed biotech rice generated by transgenesis was thus conducted, based on 1067 peer-reviewed publications, and analysed regarding inter alia their expressed genes of interest and the corresponding traits, their transformation processes and the elements composing their transgenic cassettes. From this work, the power and weakness of the standard detection system, notably used by the European enforcement laboratories, are evaluated. To strengthen this system, especially with unauthorized GMO, additional strategies are suggested. Moreover, given the growing interest for biotech rice produced by new plant breeding techniques, related challenges for their detection are discussed.

Key findings and conclusions

According to all collected information, suitable detection strategies, combining qPCR to additional technologies (e.g., DNA walking and NGS), are proposed to cover most of inventoried biotech rice. The present approach, including the data centralization to subsequently suggest appropriated detection strategies, can be extended to biotech events from different species.

VL - 52 M3 - https://doi.org/10.1016/j.tifs.2016.03.011 ER - TY - Generic T1 - Data analysis associated with the use of whole genome sequencing for subtyping of pathogenic microorganisms, Salmonella Typhimurium as a case study Y1 - 2016 A1 - Assia Saltykova A1 - Wuyts,V. A1 - Sophie Bertrand A1 - Nancy Roosens A1 - Marchal,K. A1 - Sigrid C.J. De Keersmaecker KW - a KW - an KW - analysi KW - analysis KW - application KW - approach KW - approaches KW - AS KW - at KW - bioinformatics KW - Case KW - case studies KW - Case study KW - case-study KW - Comparison KW - continue KW - Cost KW - data KW - Decline KW - detection KW - discrimination KW - ECONOMIC KW - EPIDEMIOLOGICAL KW - Genome KW - Human KW - identification KW - INFECTION KW - INFORMATION KW - IS KW - IT KW - legal KW - Life KW - microorganism KW - microorganisms KW - NGS KW - ON KW - ORIGIN KW - outbreak KW - parameters KW - pathogenic KW - Quality KW - result KW - results KW - routine KW - Salmonella KW - Salmonella enterica KW - Salmonella typhimurium KW - Science KW - situation KW - SOCIAL KW - Species KW - Still KW - study KW - Subtyping KW - subtyping of isolates KW - Surveillance KW - use KW - WGS KW - whole genome KW - whole genome sequencing AB -

As the cost of bacterial whole genome sequencing (WGS) by NGS continues to decline, its application for routine (sub)typing of pathogenic microorganisms quickly becomes a reality. NGS allows to examine relationships between different bacterial isolates of the same species at the highest possible resolution, which is essential for epidemiological surveillance and outbreak detection. However, it is still a largely unresolved question how to convert WGS data into information of sufficient quality to be reliably used for epidemiological characterization and discrimination, including the identification of a link between the origin of an infection and the human isolate in case of outbreak situations.Here, we have explored the possibilities offered by single nucleotide polymorphism (SNP)-based approach implemented in three different workflows to subtype 32

JF - Applied Bioinformatics in Life Sciences PB - NA CY - NA CP - VIB U1 - 5269 U2 - 03/17/2016-03/18/2016 ER - TY - Generic T1 - Development and implementation of a transversal NGS & bioinformatics platform at the Belgian Institute of Public Health Y1 - 2016 A1 - Kevin Vanneste A1 - Bert Bogaerts A1 - Q. Fu A1 - Raf Winand A1 - Romeu,A.G. A1 - Brown,I. A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens KW - a KW - acquisition KW - an KW - analysi KW - analysis KW - AS KW - at KW - Belgian KW - bioinformatics KW - Case KW - challenge KW - Clinical KW - contribution KW - Control KW - criteria KW - data KW - Database KW - Databases KW - Development KW - Diagnosis KW - effective KW - Emergencies KW - Emergency KW - expertise KW - genomic KW - Genomics KW - Guidelines KW - health KW - HEALTH POLICIES KW - HEALTH POLICY KW - implementation KW - Institute KW - interaction KW - IS KW - KNOWLEDGE KW - Laboratories KW - Life KW - Mass KW - Mass Spectrometry KW - method KW - NGS KW - POLICIES KW - POLICY KW - present KW - public KW - public health KW - public health genomics KW - Public-health AB -

Despite being a well-established research method, the use of NGS and bioinformatics for routine analysis in a public health setting remains a challenge. The NGS and bioinformatics platform was recently set up at the Belgian Institute of Public Health with the aim of utilizing NGS and bioinformatics for the diagnosis, surveillance, control and characterization of potentially harmful organisms; and to promote public health genomics by the effective integration of NGS and bioinformatics into clinical use and public health policy.The platform develops solutions and provides data acquisition and analysis tools to complement the WIV-ISP laboratories services; and to integrate the knowledge of genomics into public health policy. The platform has built up the capacity to both generate and analyze NGS data through an in-house Miseq and advanced bioinformatics pipelines and databases. These services are developed under a strict quality system and offered as a high-quality service platform with the aim of being adapted for routine analysis for both surveillance and emergency cases. Expertise is present in regulation and quality control by active contribution to (inter)national workgroups for the development of guidelines and criteria for NGS. Novel solutions are actively being researched and developed with the aim of supporting a proactive public health policy. Lastly, interaction with other high-throughput technologies such as mass spectrometry, are actively being investigated.

JF - Applied Bioinformatics in Life Sciences PB - NA CY - NA CP - VIB U1 - 5270 U2 - 03/17/2016-03/18/2016 ER - TY - Generic T1 - Development and implementation of a transversal NGS & bioinformatics platform at the Belgian Institute of Public Health: deployment of user-friendly pipelines for routine use Y1 - 2016 A1 - Kevin Vanneste A1 - Q. Fu A1 - Bert Bogaerts A1 - Raf Winand A1 - Romeu,A.G. A1 - Brown,I. A1 - Julien Van Braekel A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens KW - a KW - at KW - Belgian KW - bioinformatics KW - Biology KW - Computational Biology KW - conference KW - Development KW - European KW - Genomics KW - health KW - implementation KW - Institute KW - NGS KW - ON KW - public KW - public health KW - Public-health KW - routine KW - use KW - WGS JF - 15th European Conference on Computational Biology PB - VU Amsterdam, TU Delft, DTL, BioSB, UMC Utrecht CY - NA CP - VU Amsterdam, TU Delft, DTL, BioSB, UMC Utrecht U1 - 5284 U2 - 09/04-07/2016 ER - TY - Generic T1 - Development and use of non-invasive biomarkers to monitor respiratory health of young children Y1 - 2016 A1 - S. Nauwelaerts A1 - Koen De Cremer A1 - Olivier J Denis A1 - A. Bernard A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens KW - Biomarkers KW - CHILDREN KW - Development KW - health KW - Institute KW - International KW - Network KW - Respiratory KW - use KW - young JF - Institute Pasteur International Network Scientific Symposium PB - WIV-ISP CY - Paris, France CP - Institut Pasteur U1 - 593 U2 - 29 november-2 december 2016 ER - TY - Generic T1 - Development of a molecular alternative to classical microbiological subtyping methods, Salmonella phage typing as a case study Y1 - 2016 A1 - Wuyts,V. A1 - Sophie Bertrand A1 - Marchal,K. A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker ED - Johan Peeters KW - a KW - alternative KW - AS KW - Case KW - case studies KW - Case study KW - case-study KW - Development KW - method KW - methods KW - Molecular KW - report KW - Salmonella KW - study KW - Subtyping PB - WIV-ISP CY - Brussels, Belgium SN - D/2015/2505/03 U1 - 5787 ER - TY - RPRT T1 - Goal 4: Develop and implement new technologies and methods to improve WIV-ISP's responsiveness to public health threats Y1 - 2016 A1 - Myriam Sneyers A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker ED - Johan Peeters KW - AS KW - Case KW - Development KW - health KW - implement KW - improve KW - method KW - methods KW - public KW - public health KW - Public-health KW - report KW - Salmonella KW - study KW - Subtyping KW - technology KW - WIV-ISP PB - WIV-ISP CY - Brussels, Belgium SN - D/2015/2505/03 U1 - 5786 ER - TY - Generic T1 - High-tech technologies for the detection of unauthorised Genetically Modified Organisms in food and feed for safety control of the food chain T2 - Scientific report 2014-2015 Y1 - 2016 A1 - Marie-Alice Fraiture A1 - Philippe Herman A1 - Taverniers, Isabel A1 - De Loose, Marc A1 - Berben, Gilbert A1 - Nancy Roosens KW - High-tech technologies for the detection of unauthorised Genetically Modified Organisms in food and feed for safety control of the food chain JF - Scientific report 2014-2015 PB - WIV-ISP CY - Brussels, Belgium UR - / ER - TY - Generic T1 - Molecular and clinical investigation of Zika virus outbreak in New Caledonia Y1 - 2016 A1 - Dupont-Rouzeyrol,M. A1 - Gourinat,A.C. A1 - Caro,V. A1 - Calvez,E. A1 - Sevila,J. A1 - O'Connor,O. A1 - Vanhomwegen,J. A1 - Diancourt,L. A1 - Simon,O. A1 - Biron,A. A1 - Nancy Roosens A1 - Faye,O. A1 - Descloux,E. A1 - Grangeon,J.P. KW - 2007 KW - a KW - an KW - analysi KW - analysis KW - AS KW - Brazil KW - Case KW - Clinical KW - Cluster KW - Congresses KW - detection KW - Disorder KW - Disorders KW - Emergencies KW - Emergency KW - EPIDEMIOLOGICAL KW - Evolution KW - health KW - Human KW - Humans KW - INFECTION KW - International KW - investigation KW - IS KW - Malaria KW - Medicine KW - method KW - Molecular KW - neurological KW - non-invasive KW - ON KW - outbreak KW - outbreak investigation KW - pathogen KW - public KW - public health KW - Public-health KW - region KW - result KW - results KW - Rna KW - Sample KW - Samples KW - SENSITIVITY KW - serum KW - Spread KW - strain KW - time KW - urine KW - VIRUS KW - WHO KW - world KW - Zika virus AB -

Introduction: Zika virus (ZIKV) is an emerging mosquito-borne pathogen transmitted to humans byinfected Aedes mosquitoes. In 2016, WHO declared ZIKV as a Public Health Emergency ofInternational Concern regarding clusters of microcephaly cases and neurological disorders probablylinked to ZIKV infection. Before that, ZIKV emerged in the Pacific for the first time in 2007. In 2013French Polynesia (FP) experienced a large Zika outbreak. ZIKV then spread throughout the Pacificduring the following two years and reached Brazil where a major outbreak is occurring. In NewCaledonia (NC), a ZIKV outbreak occurred in 2014 and 2015 with more than 1500 cases of ZIKVconfirmed by RT-PCR.Method: However, the diagnostic of ZIKV cases was challenging due to low sensitivity of RT-PCRtechnics on serum samples. We thus explored the detection of ZIKV in non-invasive samples. Wealso investigated the molecular evolution of ZIKV in NC compared to other regions in the world.Results/Conclusion: Here, we highlighted a better sensitivity of ZIKV detection by RT-PCR in urinesamples with longer and higher presence of ZIKV RNA compared to serum. Phylogenetic analysisconfirmed the epidemiological link between FP and NC ZIKV strains. Finally results of clinicalinvestigations regarding probable neurological disorders linked to ZIKV infection will be presented.

JF - International Congress of Tropical Medicine and Malaria PB - NA CY - NA CP - ASP, ASID, IFTM U1 - 5288 U2 - 09/18-22/2016 ER - TY - JOUR T1 - A molecular approach for the rapid, selective and sensitive detection of Exophiala jeanselmei in environmental samples: development and performance assessment of a real-time PCR assay JF - Appl Microbiol Biotechnol Y1 - 2016 A1 - Libert, X A1 - Camille Chasseur A1 - Ann Packeu A1 - Bureau, F A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - Base Sequence KW - DNA Primers KW - DNA, Fungal KW - DNA, Ribosomal KW - Environmental Microbiology KW - Exophiala KW - Molecular Sequence Data KW - Mycological Typing Techniques KW - Real-Time Polymerase Chain Reaction KW - Sensitivity and Specificity AB -

Exophiala jeanselmei is an opportunistic pathogenic black yeast growing in humid environments such as water reservoirs of air-conditioning systems. Because this fungal contaminant could be vaporized into the air and subsequently cause health problems, its monitoring is recommended. Currently, this monitoring is based on culture and microscopic identification which are complex, sometimes ambiguous and time-demanding, i.e., up to 21 days. Therefore, molecular, culture-independent methods could be more advantageous for the monitoring of E. jeanselmei. In this study, we developed a SYBR®green real-time PCR assay based on the internal transcribed spacer 2 from the 18S ribosomal DNA complex for the specific detection of E. jeanselmei. The selectivity (100 %), PCR efficiency (95.5 %), dynamic range and repeatability of this qPCR assay were subsequently evaluated. The limit of detection for this qPCR assay was determined to be 1 copy of genomic DNA of E. jeanselmei. Finally, water samples collected from cooling reservoirs were analyzed using this qPCR assay to deliver a proof of concept for the molecular detection of E. jeanselmei in environmental samples. The results obtained by molecular analysis were compared with those of classical methods (i.e., culture and microscopic identification) used in routine analysis and were 100 % matching. This comparison demonstrated that this SYBR®green qPCR assay can be used as a molecular alternative for monitoring and routine investigation of samples contaminated by E. jeanselmei, while eliminating the need for culturing and thereby considerably decreasing the required analysis time to 2 days.

VL - 100 CP - 3 U1 - http://www.ncbi.nlm.nih.gov/pubmed/26615400?dopt=Abstract M3 - 10.1007/s00253-015-7175-z ER - TY - BOOK T1 - Molecular subtyping of Salmonella Typhimurium with multiplex oligonucleotide ligation-PCR (MOL-PCR) T2 - Diagnostic Bacteriology Protocols Y1 - 2016 A1 - Wuyts,V. A1 - Wesley Mattheus A1 - Nancy Roosens A1 - Marchal,K. A1 - Sophie Bertrand A1 - Sigrid C.J. De Keersmaecker ED - Bishop-Lilly,K. KW - a KW - analysi KW - analysis KW - AS KW - bacteria KW - bead suspension array KW - Biology KW - Control KW - data KW - detection KW - Diagnosis KW - disease KW - Diseases KW - Dna KW - Genetic KW - Gödel Prime Product KW - high-throughput assay KW - IS KW - Isolation KW - IT KW - Luminex KW - lysis KW - Marker KW - Markers KW - method KW - methods KW - MOL-PCR KW - Molecular KW - Molecular biology KW - molecular markers KW - molecular subtyping KW - Multiplex assay KW - ON KW - pathogen KW - PCR KW - PRODUCTS KW - protocol KW - S KW - Salmonella KW - Salmonella enterica KW - Salmonella typhimurium KW - Subtyping KW - Technique KW - Type KW - Viruses AB - A multiplex oligonucleotide ligation-PCR (MOL-PCR) assay is a valuable high-throughput technique for the detection of bacteria and viruses, for characterization of pathogens and for diagnosis of genetic diseases, as it allows to combine different types of molecular markers in a high-throughput multiplex assay. A MOL-PCR assay starts with a multiplex oligonucleotide ligation reaction for detection of the molecular marker, followed by a singleplex PCR for signal amplification and analysis of the MOL-PCR products on a Luminex platform. This last step occurs through a liquid bead suspension array in which the MOL-PCR products are hybridized to MagPlex-TAG beads.In this chapter, we describe the complete procedure for a MOL-PCR assay for subtyping of JF - Diagnostic Bacteriology Protocols PB - Humana Press CY - Totowa, New Jersey SN - 978-1-58829-594-1 U1 - 568 ER - TY - JOUR T1 - Optimized MOL-PCR for Characterization of Microbial Pathogens JF - Curr Protoc Cytom Y1 - 2016 A1 - Wuyts, Véronique A1 - Nancy Roosens A1 - Sophie Bertrand A1 - Marchal, Kathleen A1 - Sigrid C.J. De Keersmaecker KW - bacteria KW - Biomarkers KW - Biotinylation KW - DNA, Bacterial KW - Electrophoresis, Gel, Pulsed-Field KW - Fluorescent Dyes KW - Genetic Markers KW - Humans KW - Nucleic Acid Hybridization KW - Oligonucleotide Probes KW - Phycoerythrin KW - polymerase chain reaction KW - Polymorphism, Single Nucleotide KW - Salmonella typhimurium KW - Streptavidin KW - Temperature AB -

Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation-PCR (MOL-PCR) optimized for characterization of microbial pathogens. With MOL-PCR, different types of markers, like unique sequences, single-nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex-TAG beads for readout on a Luminex platform after fluorescent staining. The current protocol describes the MOL-PCR, as well as methods for DNA isolation, probe design, and data interpretation and it is based on an optimized MOL-PCR assay for subtyping of Salmonella Typhimurium.

VL - 75 U1 - http://www.ncbi.nlm.nih.gov/pubmed/26742655?dopt=Abstract M3 - 10.1002/0471142956.cy1315s75 ER - TY - JOUR T1 - Statistical framework for detection of Genetically Modified Organisms based on Next Generation Sequencing JF - Food Chemistry Y1 - 2016 A1 - Willems, Sander A1 - Marie-Alice Fraiture A1 - Deforce, Dieter A1 - Sigrid C.J. De Keersmaecker A1 - Philippe Herman A1 - De Loose, Marc A1 - Ruttink, Tom A1 - Van Nieuwerburgh, Filip A1 - Nancy Roosens KW - bioinformatics KW - detection KW - GM rice KW - GMO KW - NGS KW - Processed food KW - Statistical framework AB -

Because the number and diversity of genetically modified (GM) crops has significantly increased, their analysis based on real-time PCR (qPCR) methods is becoming increasingly complex and laborious. While several pioneers already investigated Next Generation Sequencing (NGS) as an alternative to qPCR, its practical use has not been assessed for routine analysis. In this study a statistical framework was developed to predict the number of NGS reads needed to detect transgene sequences, to prove their integration into the host genome and to identify the specific transgene event in a sample with known composition. This framework was validated by applying it to experimental data from food matrices composed of pure GM rice, processed GM rice (noodles) or a 10% GM/non-GM rice mixture, revealing some influential factors. Finally, feasibility of NGS for routine analysis of GM crops was investigated by applying the framework to samples commonly encountered in routine analysis of GM crops.

VL - 192 CP - 1 M3 - https://doi.org/10.1016/j.foodchem.2015.07.074 ER - TY - Generic T1 - Whole genome sequencing to help enforcement laboratories for the development of detection method for an EU-unauthorized genetically modified Bacillus subtilis overproducing riboflavin Y1 - 2016 A1 - Sigrid C.J. De Keersmaecker A1 - N. Papazova A1 - Nancy Roosens KW - abstract KW - an KW - Bacillus KW - Bacillus subtilis KW - detection KW - detection method KW - Development KW - genetically KW - Genetically modified KW - Genome KW - GMO KW - GMO detection KW - Help KW - Laboratories KW - method KW - riboflavin KW - WGS KW - whole genome KW - whole genome sequencing AB - no abstract JF - LabInfo VL - 15 CP - Jul-16 U1 - 5282 ER - TY - Generic T1 - Arbo-VIRTUESS: Evaluation of the use of non-invasive tests for early screening and survey of arboviruses Y1 - 2015 A1 - Sylvia Broeders A1 - Sigrid C.J. De Keersmaecker A1 - Vanessa Suin A1 - Steven Van Gucht A1 - Pol,M. A1 - Gourinat,A.C. A1 - Biron,A. A1 - Dupont-Rouzeyrol,M. A1 - Vanhomwegen,J. A1 - Manuguerra,J.C. A1 - Berlioz-Arthaud,A. A1 - Enfissi,A. A1 - Matheus,S A1 - Rousset,D. A1 - Nancy Roosens KW - Arbo-virtuess KW - EVALUATION KW - International KW - non-invasive KW - SCREENING KW - survey KW - symposium KW - Test KW - tests KW - use JF - International Scientific Symposium PB - NA CY - NA CP - Institut Pasteur International Network U1 - 5224 U2 - 14-16/10/2015 ER - TY - JOUR T1 - Current and new approaches in GMO detection: challenges and solutions JF - BioMed Research International Y1 - 2015 A1 - Marie-Alice Fraiture A1 - Philippe Herman A1 - Taverniers, Isabel A1 - De Loose, Marc A1 - Deforce, Dieter A1 - Nancy Roosens KW - Current and New Approaches in GMO Detection: Challenges and Solutions AB -

In many countries, genetically modified organisms (GMO) legislations have been established in order to guarantee the traceability of food/feed products on the market and to protect the consumer freedom of choice. Therefore, several GMO detection strategies, mainly based on DNA, have been developed to implement these legislations. Due to its numerous advantages, the quantitative PCR (qPCR) is the method of choice for the enforcement laboratories in GMO routine analysis. However, given the increasing number and diversity of GMO developed and put on the market around the world, some technical hurdles could be encountered with the qPCR technology, mainly owing to its inherent properties. To address these challenges, alternative GMO detection methods have been developed, allowing faster detections of single GM target (e.g., loop-mediated isothermal amplification), simultaneous detections of multiple GM targets (e.g., PCR capillary gel electrophoresis, microarray, and Luminex), more accurate quantification of GM targets (e.g., digital PCR), or characterization of partially known (e.g., DNA walking and Next Generation Sequencing (NGS)) or unknown (e.g., NGS) GMO. The benefits and drawbacks of these methods are discussed in this review.

VL - 2015 CP - 392872 M3 - http://dx.doi.org/10.1155/2015/392872 ER - TY - JOUR T1 - Development and performance assessment of a qualitative SYBR® green real-time PCR assay for the detection of Aspergillus versicolor in indoor air. JF - Appl Microbiol Biotechnol Y1 - 2015 A1 - Libert, X A1 - Camille Chasseur A1 - Bladt, S A1 - Ann Packeu A1 - Bureau, F A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - Air Microbiology KW - Air Pollution, Indoor KW - Aspergillus KW - DNA, Fungal KW - DNA, Ribosomal Spacer KW - Humans KW - Organic Chemicals KW - Real-Time Polymerase Chain Reaction KW - Reproducibility of Results KW - Sensitivity and Specificity KW - Staining and Labeling AB -

Currently, contamination of indoor environment by fungi and molds is considered as a public health problem. The monitoring of indoor airborne fungal contamination is a common tool to help understanding the link between fungi in houses and respiratory problems. Classical analytical monitoring methods, based on cultivation and microscopic identification, depend on the growth of the fungi. Consequently, they are biased by difficulties to grow some species on certain culture media and under certain conditions or by noncultivable or dead fungi that can consequently not be identified. However, they could have an impact on human health as they might be allergenic. Since molecular methods do not require a culture step, they seem an excellent alternative for the monitoring of indoor fungal contaminations. As a case study, we developed a SYBR® green real-time PCR-based assay for the specific detection and identification of Aspergillus versicolor, which is frequently observed in indoor environment and known to be allergenic. The developed primers amplify a short region of the internal transcribed spacer 1 from the 18S ribosomal DNA complex. Subsequently, the performance of this quantitative polymerase chain reaction (qPCR) method was assessed using specific criteria, including an evaluation of the selectivity, PCR efficiency, dynamic range, and repeatability. The limit of detection was determined to be 1 or 2 copies of genomic DNA of A. versicolor. In order to demonstrate that this SYBR® green qPCR assay is a valuable alternative for monitoring indoor fungal contamination with A. versicolor, environmental samples collected in contaminated houses were analyzed and the results were compared to the ones obtained with the traditional methods.

VL - 99 CP - 17 U1 - http://www.ncbi.nlm.nih.gov/pubmed/26184975?dopt=Abstract M3 - 10.1007/s00253-015-6785-9 ER - TY - JOUR T1 - Erratum to: Development and performance assessment of a qualitative SYBR® green real-time PCR assay for the detection of Aspergillus versicolor in indoor air. JF - Appl Microbiol Biotechnol Y1 - 2015 A1 - Libert, X A1 - Camille Chasseur A1 - Bladt, S A1 - Ann Packeu A1 - Bureau, F A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker VL - 99 CP - 19 U1 - http://www.ncbi.nlm.nih.gov/pubmed/26293339?dopt=Abstract M3 - 10.1007/s00253-015-6932-3 ER - TY - RPRT T1 - Evaluation of a DNA extraction kit for the use of saliva as a non-invasive source of human DNA biomarkers in support of public health genomics. Y1 - 2015 A1 - Ballot,C A1 - De Wit,V A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - a KW - AS KW - Biomarkers KW - Dna KW - DNA biomarkers KW - EVALUATION KW - genomic KW - health KW - Human KW - non-invasive KW - public KW - public health KW - Public-health KW - Saliva KW - use PB - Koninklijke Bibliotheek België/Bibliothèque Royale de Belgique CY - Brussels, Belgium SN - D/2015/2505/64. U1 - 5242 ER - TY - Generic T1 - Evaluation of whole genome sequencing for routine subtyping of Salmonella Typhimurium for surveillance and outbreak investigation Y1 - 2015 A1 - Wuyts,V. A1 - Sophie Bertrand A1 - Wesley Mattheus A1 - Nancy Roosens A1 - Marchal,K. A1 - Sigrid C.J. De Keersmaecker KW - a KW - additional KW - an KW - analysi KW - analysis KW - Antibiotic KW - Antibiotic resistance KW - AS KW - aspects KW - at KW - Belgian KW - Case KW - data KW - Diagnosis KW - disease KW - Diseases KW - Electrophoresis KW - epidemiology KW - Evaluating KW - EVALUATION KW - Genome KW - Genotype KW - Human KW - identify KW - INFECTION KW - Infectious KW - Infectious diseases KW - investigation KW - IS KW - LEVEL KW - method KW - national KW - Next-generation sequencing KW - NGS KW - ORIGIN KW - outbreak KW - Phenotype KW - resistance KW - routine KW - S KW - Salmonella KW - Salmonella enterica KW - Salmonella typhimurium KW - Shigella KW - situation KW - Species KW - study KW - Subtyping KW - Surveillance KW - Technique KW - technology KW - whole genome KW - whole genome sequencing AB -

Characterisation of bacterial isolates beyond the species and subspecies level, i.e. subtyping, is necessary for surveillance of infectious diseases and is crucial to identify a link between the origin of an infection and the human isolate in case of outbreak situations.At the Belgian National Reference Centre (NRC) for

JF - Revolutionizing Next-Generation Sequencing: Tools and Technologies PB - NA CY - NA CP - VIB U1 - 5086 U2 - 15-16/01/2015 ER - TY - JOUR T1 - Fast and discriminative CoSYPS detection system of viable Salmonella spp. and Listeria spp. in carcass swab samples. JF - Int J Food Microbiol Y1 - 2015 A1 - Barbau-Piednoir, Elodie A1 - N Botteldoorn A1 - Mahillon, Jacques A1 - Katelijne Dierick A1 - Nancy Roosens KW - Animals KW - Cattle KW - Food Microbiology KW - Listeria KW - Meat KW - Real-Time Polymerase Chain Reaction KW - Salmonella AB -

In this study, the complete CoSYPS Path Food workflow including all steps, namely swab sample enrichment, SYBR®Green qPCR detection of Salmonella spp. and Listeria spp., isolation and confirmation of the detected strain, was validated on beef carcass swabs. To perform the validation, the results of the complete workflow were compared, according to the ISO 16140:2003, with the ISO reference methods for detection, isolation and confirmation of Listeria monocytogenes and Salmonella spp. The results showed that the relative level of detection and the limit of detection of the complete workflow and ISO reference methods are in a range from 2 to 16 CFU/swab for both bacteria. The relative specificity, sensitivity and accuracy identified during this validation were all 100% since the results obtained with the complete CoSYPS Path Food workflow and the ISO reference methods were identical (Cohen's kappa index=1.00). In addition the complete CoSYPS Path Food workflow is able to provide detection results (negative or presumptive positive) in half the time needed as for the ISO reference methods. These results demonstrate that the performance of the complete CoSYPS Path Food workflow is not only comparable to the ISO reference methods but also provides a faster response for the verification of beef carcasses before commercial distribution.

VL - 192 U1 - http://www.ncbi.nlm.nih.gov/pubmed/25440553?dopt=Abstract M3 - 10.1016/j.ijfoodmicro.2014.09.018 ER - TY - JOUR T1 - Genome Sequence of EU-Unauthorized Genetically Modified Bacillus subtilis Strain 2014-3557 Overproducing Riboflavin, Isolated from a Vitamin B2 80% Feed Additive. JF - Genome Announc Y1 - 2015 A1 - Barbau-Piednoir, Elodie A1 - Sigrid C.J. De Keersmaecker A1 - Wuyts, Véronique A1 - Gau, Céline A1 - Pirovano, Walter A1 - Costessi, Adalberto A1 - Philipp, Patrick A1 - Nancy Roosens AB -

This paper announces the genome sequence and annotation of the genetically modified (GM) Bacillus subtilis strain 2014-3557 overproducing riboflavin (vitamin B2). This GM-strain is unauthorized in the European Union. Nevertheless, it has been isolated from a lot of vitamin B2 (riboflavin) 80% feed grade imported to Europe from China.

VL - 3 CP - 2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/25858836?dopt=Abstract M3 - 10.1128/genomeA.00214-15 ER - TY - Generic T1 - GMM detection in food & feed : B. subtilis as case study Y1 - 2015 A1 - Nancy Roosens KW - AS KW - Case KW - case studies KW - Case study KW - case-study KW - detection KW - feed KW - food KW - GMM KW - study KW - Workshop JF - NRL-GMO Theoretical workshop PB - NA CY - NA CP - NRL-GMO/FAVV U1 - 5233 U2 - 04/06/2015 ER - TY - JOUR T1 - Guidelines for optimisation of a multiplex oligonucleotide ligation-PCR for characterisation of microbial pathogens in a microsphere suspension array. JF - Biomed Res Int Y1 - 2015 A1 - Wuyts, Véronique A1 - Nancy Roosens A1 - Sophie Bertrand A1 - Marchal, Kathleen A1 - Sigrid C.J. De Keersmaecker KW - DNA Primers KW - DNA, Bacterial KW - Humans KW - Microspheres KW - Multiplex Polymerase Chain Reaction KW - Oligonucleotide Probes KW - Salmonella typhimurium AB -

With multiplex oligonucleotide ligation-PCR (MOL-PCR) different molecular markers can be simultaneously analysed in a single assay and high levels of multiplexing can be achieved in high-throughput format. As such, MOL-PCR is a convenient solution for microbial detection and identification assays where many markers should be analysed, including for routine further characterisation of an identified microbial pathogenic isolate. For an assay aimed at routine use, optimisation in terms of differentiation between positive and negative results and of cost and effort is indispensable. As MOL-PCR includes a multiplex ligation step, followed by a singleplex PCR and analysis with microspheres on a Luminex device, several parameters are accessible for optimisation. Although MOL-PCR performance may be influenced by the markers used in the assay and the targeted bacterial species, evaluation of the method of DNA isolation, the probe concentration, the amount of microspheres, and the concentration of reporter dye is advisable in the development of any MOL-PCR assay. Therefore, we here describe our observations made during the optimisation of a 20-plex MOL-PCR assay for subtyping of Salmonella Typhimurium with the aim to provide a possible workflow as guidance for the development and optimisation of a MOL-PCR assay for the characterisation of other microbial pathogens.

VL - 2015 U1 - http://www.ncbi.nlm.nih.gov/pubmed/25705689?dopt=Abstract M3 - 10.1155/2015/790170 ER - TY - JOUR T1 - Integrated DNA walking system to characterize a broad spectrum of GMOs in food/feed matrices. JF - BMC Biotechnology Y1 - 2015 A1 - Marie-Alice Fraiture A1 - Philippe Herman A1 - Lefèvre, Loic A1 - Taverniers, Isabel A1 - De Loose, Marc A1 - Deforce, Dieter A1 - Nancy Roosens KW - Integrated DNA walking system to characterize a broad spectrum of GMOs in food/feed matrices AB -

Background

In order to provide a system fully integrated with qPCR screening, usually used in GMO routine analysis, as well as being able to detect, characterize and identify a broad spectrum of GMOs in food/feed matrices, two bidirectional DNA walking methods targeting p35S or tNOS, the most common transgenic elements found in GM crops, were developed. These newly developed DNA walking methods are completing the previously implemented DNA walking method targeting the t35S pCAMBIA element.

Methods

Food/feed matrices containing transgenic crops (Bt rice or MON863 maize) were analysed using the integrated DNA walking system.

Results

First, the newly developed DNA walking methods, anchored on the sequences used for the p35S or tNOS qPCR screening, were tested on Bt rice that contains these two transgenic elements. Second, the methods were assessed on a maize sample containing a low amount of the GM MON863 event, representing a more complex matrix in terms of genome size and sensitivity. Finally, to illustrate its applicability in GMO routine analysis by enforcement laboratories, the entire workflow of the integrated strategy, including qPCR screening to detect the potential presence of GMOs and the subsequent DNA walking methods to characterize and identify the detected GMOs, was applied on a GeMMA Scheme Proficiency Test matrix. Via the characterization of the transgene flanking region between the transgenic cassette and the plant genome as well as of a part of the transgenic cassette, the presence of GMOs was properly confirmed or infirmed in all tested samples.

Conclusion

Due to their simple procedure and their short time-frame to get results, the developed DNA walking methods proposed here can be easily implemented in GMO routine analysis by the enforcement laboratories. In providing crucial information about the transgene flanking regions and/or the transgenic cassettes, this DNA walking strategy is a key molecular tool to prove the presence of GMOs in any given food/feed matrix.

VL - 15 CP - 76 M3 - https://doi.org/10.1186/s12896-015-0191-3 ER - TY - JOUR T1 - A multiplex oligonucleotide ligation-PCR as a complementary tool for subtyping of Salmonella Typhimurium JF - Appl Microbiol Biotechnol Y1 - 2015 A1 - Wuyts,V. A1 - Wesley Mattheus A1 - Nancy Roosens A1 - Marchal,K. A1 - Sophie Bertrand A1 - Sigrid C.J. De Keersmaecker KW - analysis KW - Antibiotic KW - Antibiotic resistance KW - AS KW - cause KW - causes KW - Concordance KW - data KW - detection KW - EPIDEMIOLOGICAL KW - gene KW - Genes KW - genomic KW - INFECTION KW - infections KW - International KW - investigation KW - IS KW - IT KW - Laboratories KW - LEVEL KW - Luminex KW - Marker KW - Markers KW - method KW - microsphere array KW - MOL-PCR KW - Molecular KW - molecular markers KW - Network KW - Objective KW - observed KW - ON KW - outbreak KW - outbreaks KW - resistance KW - result KW - results KW - S KW - Salmonella KW - Salmonella enterica KW - Salmonella typhimurium KW - stability KW - Subtyping KW - Surveillance KW - Technique KW - Type KW - use KW - VALIDATION AB -

Subtyping below the serovar level is essential for surveillance and outbreak detection and investigation of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant 1,4,[5],12:i:- (S. 1,4,[5],12:i:-), frequent causes of foodborne infections. In an attempt to overcome the intrinsic shortcomings of currently used subtyping techniques, a multiplex oligonucleotide ligation-PCR (MOL-PCR) assay was developed which combines different types of molecular markers in a high-throughput microsphere suspension array. The 52 molecular markers include prophage genes, amplified fragment length polymorphism (AFLP) elements, Salmonella genomic island 1 (SGI1), allantoinase gene allB, MLVA locus STTR10, antibiotic resistance genes, single nucleotide polymorphisms (SNPs) and phase 2 flagellar gene fljB. The in vitro stability of these markers was confirmed in a serial passage experiment. The validation of the MOL-PCR assay for subtyping of S. Typhimurium and S. 1,4,[5],12:i:- on 519 isolates shows that the method is rapid, reproducible, flexible, accessible, easy to use and relatively inexpensive. Additionally, a 100 % typeability and a discriminatory power equivalent to that of phage typing were observed, and epidemiological concordance was assessed on isolates of 2 different outbreaks. Furthermore, a data analysis method is provided so that the MOL-PCR assay allows for objective, computerised data analysis and data interpretation of which the results can be easily exchanged between different laboratories in an international surveillance network.

VL - 99 SN - 0175-7598 CP - 19 U1 - 5212 M3 - 10.1007/s00253-015-6831-7 ER - TY - JOUR T1 - New trait-specific qualitative SYBR®Green qPCR methods to expand the panel of GMO screening methods used in the CoSYPS JF - European Food Research and Technology Y1 - 2015 A1 - Sylvia Broeders A1 - Marie-Alice Fraiture A1 - Els Vandermassen A1 - Delvoye, Maud A1 - Barbau-Piednoir, Elodie A1 - Lievens, Antoon A1 - Nancy Roosens KW - New qualitative trait-specific SYBR®Green qPCR methods to expand the panel of GMO screening methods used in the CoSYPS AB -

Since 2011, a new Commission Regulation (EU/619/2011) defines that laboratories testing for genetically modified organisms (GMO) need to be able to detect also genetically modified (GM) events pending for authorisation. This, in addition to the fact that the number of GM events authorised in the European Union (EU) that need to be identified multiplies rapidly and that the detection of unauthorised GMO becomes more important, led to the development of a time and cost-effective screening approach. Moreover, the GM elements that are utilised in the transgenic inserts also become increasingly diverse. Consequently, the screening approaches have to be updated to enable full coverage and better discrimination of all these events. To respond to this need, two new qualitative SYBR®Green real-time PCR (qPCR) methods were developed and in-house validated: one method is element-specific and targets the Cry3Bb trait, and the other one is a construct-specific method detecting the gat-tpinII junction. Method acceptance parameters such as the sensitivity, specificity and repeatability were assessed as well as the robustness of the methods. Additionally, the reproducibility was evaluated by transferring the methods to a second laboratory. Both methods allow a specific, sensitive and repeatable detection of the respective targets in food and feed samples and can be easily applied in a routine laboratory. Moreover, they can be used together with previously validated SYBR®Green qPCR methods to expand the panel of screening methods. This allows an extended coverage of the GM events authorised in the EU and adds discriminative power to the screening phase.

VL - 241 CP - 2 M3 - https://doi.org/10.1007/s00217-015-2454-6 ER - TY - Generic T1 - Next Generation Sequencing to identify GMO in food and feed products Y1 - 2015 A1 - Willems, Sander A1 - Marie-Alice Fraiture A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens KW - Next Generation Sequencing to identify GMO in food and feed products JF - LabInfo PB - AFSCA CY - Belgium ER - TY - JOUR T1 - Next-generation sequencing as a tool for the molecular characterisation and risk assessment of genetically modified plants: Added value or not? JF - Trends Food Sci. Technol. Y1 - 2015 A1 - Katia Pauwels A1 - Sigrid C.J. De Keersmaecker A1 - Adinda De Schrijver A1 - P. du Jardin A1 - Nancy Roosens A1 - Philippe Herman KW - Genetically modified organism (GMO) KW - Genetically modified plants (GMP) KW - Next-generation sequencing (NGS) KW - Risk assessment Molecular characterisation AB -

BackgroundLegislations and international organizations provide a framework to ensure proper risk assessment of Genetically Modified Organisms (GMO). With regard to the deliberate release of GMO as food or feed, applications for Genetically Modified Plants (GMP) typically contain data for the molecular characterisation at the nucleic acid level based on Southern blot and polymerase chain reaction analysis in combination with Sanger sequencing. Along with the diverse range of applications of next-generation sequencing (NGS) in genomic research, some recent research projects and product developers explored the use of NGS as an alternative tool for meeting the data requirements for the molecular characterisation of GMPs in view of their risk assessment.Scope and approachBy means of a literature survey and information collected through the organisation of an international workshop, we investigated whether NGS can replace and/or complement the currently used techniques for molecular characterisation of GMP taking into account the possibilities and current bottlenecks of NGS technologies and recent developments in molecular breeding.Key findings and conclusionsWe conclude that although NGS might present clear advantages for product developers, NGS currently does not always offer a significant added value with respect to the risk assessment of GMPs. However, the approaches used so far may soon be further challenged by the fast evolution in NGS technologies and also by the recent developments in molecular breeding of plants. We postulate that setting up a common workflow for the generation of relevant and interpretable data by NGS would facilitate a scientifically sound assessment of GMPs.

VL - 45 SN - 0924-2244 CP - 2 U1 - 5215 M3 - 10.1016/j.tifs.2015.07.009 ER - TY - Generic T1 - Progress Report Advisory Group on Selection of Methods for Validation Y1 - 2015 A1 - Nancy Roosens KW - Group KW - method KW - methods KW - ON KW - progress report KW - report KW - Selection KW - VALIDATION JF - 18th NRL-GMO Steering Committee PB - NA CY - NA CP - NRL-GMO/FAV U1 - 5229 U2 - 24/03/2015 ER - TY - Generic T1 - RASFF GM Bacillus subtilis overproducing vitamine B2 Y1 - 2015 A1 - Nancy Roosens KW - Bacillus KW - Bacillus subtilis KW - GM JF - 18th NRL-GMO Steering Committee PB - NA CY - NA CP - NRL-GMO/FAVV U1 - 5228 U2 - 24/03/2015 ER - TY - Generic T1 - UGM characterization using DNA walking strategy Y1 - 2015 A1 - Marie-Alice Fraiture A1 - Philippe Herman A1 - N. Papazova A1 - Nancy Roosens KW - abstract KW - Dna KW - DNA walking KW - Strategies KW - Strategy KW - UGM KW - walking AB - No abstract JF - LabInfo VL - 14 U1 - 5265 ER - TY - JOUR T1 - Use of next generation sequencing data to develop a qPCR method for specific detection of EU-unauthorized genetically modified Bacillus subtilis overproducing riboflavin. JF - BMC Biotechnol Y1 - 2015 A1 - Barbau-Piednoir, Elodie A1 - Sigrid C.J. De Keersmaecker A1 - Delvoye, Maud A1 - Gau, Céline A1 - Philipp, Patrick A1 - Nancy Roosens KW - Bacillus subtilis KW - genetic engineering KW - High-Throughput Nucleotide Sequencing KW - Organisms, Genetically Modified KW - Real-Time Polymerase Chain Reaction KW - riboflavin KW - Sensitivity and Specificity KW - Sequence Analysis, DNA AB -

BACKGROUND: Recently, the presence of an unauthorized genetically modified (GM) Bacillus subtilis bacterium overproducing vitamin B2 in a feed additive was notified by the Rapid Alert System for Food and Feed (RASFF). This has demonstrated that a contamination by a GM micro-organism (GMM) may occur in feed additives and has confronted for the first time,the enforcement laboratories with this type of RASFF. As no sequence information of this GMM nor any specific detection or identification method was available, Next GenerationSequencing (NGS) was used to generate sequence information. However, NGS data analysis often requires appropriate tools, involving bioinformatics expertise which is not alwayspresent in the average enforcement laboratory. This hampers the use of this technology to rapidly obtain critical sequence information in order to be able to develop a specific qPCRdetection method.

METHODS: Data generated by NGS were exploited using a simple BLAST approach. A TaqMan® qPCR method was developed and tested on isolated bacterial strains and on the feed additive directly.

RESULTS: In this study, a very simple strategy based on the common BLAST tools that can be used by any enforcement lab without profound bioinformatics expertise, was successfully used toanalyse the B. subtilis data generated by NGS. The results were used to design and assess a new TaqMan® qPCR method, specifically detecting this GM vitamin B2 overproducing bacterium. The method complies with EU critical performance parameters for specificity, sensitivity, PCR efficiency and repeatability. The VitB2-UGM method also could detect the B. subtilis strain in genomic DNA extracted from the feed additive, without prior culturing step.

CONCLUSIONS: The proposed method, provides a crucial tool for specifically and rapidly identifying this unauthorized GM bacterium in food and feed additives by enforcement laboratories. Moreover, this work can be seen as a case study to substantiate how the use of NGS data can offer an added value to easily gain access to sequence information needed to develop qPCR methods to detect unknown andunauthorized GMO in food and feed.

VL - 15 U1 - http://www.ncbi.nlm.nih.gov/pubmed/26559764?dopt=Abstract M3 - 10.1186/s12896-015-0216-y ER - TY - Generic T1 - The usefulness of whole genome sequencing for outbreak investigation of food pathogens, Salmonella Enteritidis as a case study Y1 - 2015 A1 - Wuyts,V. A1 - Sarah Denayer A1 - Nancy Roosens A1 - Wesley Mattheus A1 - Sophie Bertrand A1 - Marchal,K. A1 - Katelijne Dierick A1 - Sigrid C.J. De Keersmaecker KW - a KW - abstract KW - AS KW - Case KW - case studies KW - Case study KW - case-study KW - food KW - Genome KW - investigation KW - outbreak KW - pathogen KW - Salmonella KW - Salmonella enteritidis KW - study KW - whole genome KW - whole genome sequencing AB -

No abstract

JF - LabInfo VL - submitted U1 - 5211 ER - TY - Generic T1 - User-friendly WGS analysis of Salmonella Enteritidis PT4 outbreaks Y1 - 2015 A1 - Wuyts,V. A1 - Sarah Denayer A1 - Nancy Roosens A1 - Wesley Mattheus A1 - Sophie Bertrand A1 - Marchal,K. A1 - Katelijne Dierick A1 - Sigrid C.J. De Keersmaecker ED - BSFM KW - analysi KW - analysis KW - conference KW - food KW - Food Microbiology KW - microbiology KW - ON KW - outbreak KW - outbreaks KW - Salmonella KW - Salmonella enteritidis KW - WGS KW - whole genome sequencing U1 - 5222 ER - TY - JOUR T1 - Validation of a sensitive DNA walking strategy to characterise unauthorised GMOs using model food matrices mimicking common rice products JF - Food Chemistry Y1 - 2015 A1 - Marie-Alice Fraiture A1 - Philippe Herman A1 - Taverniers, Isabel A1 - De Loose, Marc A1 - Van Nieuwerburgh, Filip A1 - Deforce, Dieter A1 - Nancy Roosens KW - detection KW - DNA walking KW - Rice food KW - SENSITIVITY KW - Transgene flanking region KW - Unauthorised GMO AB -

To identify unauthorised GMOs in food and feed matrices, an integrated approach has recently been developed targeting pCAMBIA family vectors, highly present in transgenic plants. Their presence is first assessed by qPCR screening and is subsequently confirmed by characterising the transgene flanking regions, using DNA walking. Here, the DNA walking performance has been thoroughly tested for the first time, regarding the targeted DNA quality and quantity. Several assays, on model food matrices mimicking common rice products, have allowed to determine the limit of detection as well as the potential effects of food mixture and processing. This detection system allows the identification of transgenic insertions as low as 10 HGEs and was not affected by the presence of untargeted DNA. Moreover, despite the clear impact of food processing on DNA quality, this method was able to cope with degraded DNA. Given its specificity, sensitivity, reliability, applicability and practicability, the proposed approach is a key detection tool, easily implementable in enforcement laboratories.

VL - 173 M3 - https://doi.org/10.1016/j.foodchem.2014.09.148 ER - TY - JOUR T1 - Whole Genome Sequence Analysis of Salmonella Enteritidis PT4 Outbreaks from a National Reference Laboratory's Viewpoint. JF - PLoS Curr Y1 - 2015 A1 - Wuyts, Véronique A1 - Sarah Denayer A1 - Nancy Roosens A1 - Wesley Mattheus A1 - Sophie Bertrand A1 - Marchal, Kathleen A1 - Katelijne Dierick A1 - Sigrid C.J. De Keersmaecker KW - a KW - additional KW - ALL KW - an KW - analysi KW - analysis KW - Antibiotic KW - Antibiotic resistance KW - Antimicrobial KW - article KW - AS KW - at KW - Belgian KW - Belgium KW - bioinformatics KW - Biology KW - Biotechnology KW - Brussels KW - Case KW - case studies KW - Case study KW - case-study KW - Common KW - data KW - Discussion KW - disease KW - Diseases KW - electronic KW - epidemiology KW - expertise KW - food KW - foodborne outbreaks KW - future KW - gene KW - Genetic KW - genetics KW - Genome KW - genomic KW - Genomics KW - health KW - Human KW - Infectious KW - Infectious diseases KW - INFORMATION KW - Institute KW - investigation KW - IS KW - IT KW - journal KW - Laboratories KW - method KW - methods KW - Molecular KW - Molecular biology KW - national KW - ON KW - ORIGIN KW - outbreak KW - outbreaks KW - pathogen KW - period KW - Phage type KW - plant KW - profile KW - public KW - public health KW - Public-health KW - Questionnaire KW - resistance KW - result KW - results KW - routine KW - routine laboratory KW - S KW - Salmonella KW - Salmonella enterica KW - Salmonella enteritidis KW - Sequence Analysis KW - Serotyping KW - Service KW - Shigella KW - SOFTWARE KW - STANDARD KW - study KW - Subtyping KW - System KW - Systems KW - technology KW - TESTING KW - time KW - Type KW - Universities KW - university KW - whole KW - whole genome AB -

INTRODUCTION: In April and May 2014, two suspected egg-related outbreaks of Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) were investigated by the Belgian National Reference Laboratory of Foodborne Outbreaks. Both the suspected food and human isolates being available, and this for both outbreaks, made these the ideal case study for a retrospective whole genome sequencing (WGS) analysis with the goal to investigate the feasibility of this technology for outbreak investigation by a National Reference Laboratory or National Reference Centre without thorough bioinformatics expertise.

METHODS: The two outbreaks were originally investigated epidemiologically with a standard questionnaire and with serotyping, phage typing, multiple-locus variable-number of tandem repeats analysis (MLVA) and antimicrobial susceptibility testing as classical microbiological methods. Retrospectively, WGS of six outbreak isolates was done on an Illumina HiSeq. Analysis of the WGS data was performed with currently available, user-friendly software and tools, namely CLC Genomics Workbench, the tools available on the server of the Center for Genomic Epidemiology and BLAST Ring Image Generator (BRIG).

RESULTS: To all collected human and food outbreak isolates, classical microbiological investigation assigned phage type PT4 (variant phage type PT4a for one human isolate) and MLVA profile 3-10-5-4-1, both of which are common for human isolates in Belgium. The WGS analysis confirmed the link between food and human isolates for each of the outbreaks and clearly discriminated between the two outbreaks occurring in a same time period, thereby suggesting a non-common source of contamination. Also, an additional plasmid carrying an antibiotic resistance gene was discovered in the human isolate with the variant phage type PT4a.

DISCUSSION: For the two investigated outbreaks occurring at geographically separated locations, the gold standard classical microbiological subtyping methods were not sufficiently discriminative to distinguish between or assign a common origin of contamination for the two outbreaks, while WGS analysis could do so. This case study demonstrated the added value of WGS for outbreak investigations by confirming and/or discriminating food and human isolates between and within outbreaks. It also proved the feasibility of WGS as complementary or even future replacing (sub)typing method for the average routine laboratory.

VL - 7 U1 - http://www.ncbi.nlm.nih.gov/pubmed/26468422?dopt=Abstract M3 - 10.1371/currents.outbreaks.aa5372d90826e6cb0136ff66bb7a62fc ER - TY - Generic T1 - AG SMV: progress report Y1 - 2014 A1 - Nancy Roosens KW - AG SMV KW - ENGL JF - 27th ENGL Steering Committee Meeting PB - NA CY - NA CP - JRC-IHCP U1 - 4890 U2 - 15/09/2014 ER - TY - RPRT T1 - Annual report of the GMO lab concerning the validation dossiers for identification of Genetically Modified Organisms (GMO): period 01/09/2013-30/08/2014. Y1 - 2014 A1 - N. Papazova A1 - Sylvia Broeders A1 - Els Vandermassen A1 - Delvoye,M. A1 - D. Van Geel A1 - Sciacqua,M. A1 - Nancy Roosens A1 - Anne-Marie Vanherle KW - annual report KW - genetically KW - Genetically modified KW - Genetically modified organism KW - Genetically modified organisms KW - GMO KW - identification KW - period KW - report KW - VALIDATION PB - WIV-ISP CY - Brussels, Belgium SN - D/2014/2505/41 U1 - 38831 ER - TY - Generic T1 - Belgian NRL-GMO inter-laboratory testing of combinatory sybr®green pcr screening (CoSYPS)4860 Y1 - 2014 A1 - N. Papazova A1 - Sylvia Broeders A1 - Nancy Roosens A1 - Jansens,E. A1 - Berben,G. A1 - Isabel Taverniers A1 - M. De Loose KW - / KW - Belgian KW - combinatory KW - CoSYPS KW - PCR KW - SCREENING KW - TESTING AB - / JF - LabInfo VL - 12 U1 - 38830 ER - TY - Generic T1 - Benefits of genomics in the risk assessment of genetically modified plants for human consumption Y1 - 2014 A1 - Willems,S. A1 - Sigrid C.J. De Keersmaecker A1 - Philippe Herman A1 - Nancy Roosens ED - Global Engage KW - a KW - analysi KW - analysis KW - application KW - applications KW - approach KW - approaches KW - Area KW - Areas KW - AS KW - assessment KW - Benefit KW - benefits KW - Combination KW - Congresses KW - CONSUMPTION KW - crops KW - Dna KW - effect KW - effects KW - Europe KW - Genetic KW - genetic modification KW - genetically KW - Genetically modified KW - Genetically modified organism KW - genetically modified plant KW - Genetically modified plants KW - genomic KW - Genomics KW - GMO KW - GMOs KW - Human KW - identification KW - Impact KW - IS KW - Molecular KW - Molecular Characterisation KW - Next-generation sequencing KW - NGS KW - ON KW - PCR KW - plant KW - Plants KW - polymerase chain reaction KW - Project KW - RANGE KW - region KW - Regulatory KW - Research KW - result KW - risk KW - Risk Assessment KW - SAFETY KW - Safety assessment KW - STANDARD KW - Transgenic KW - unintended effects KW - use AB - Within the Genetically Modified Organism (GMO) regulatory framework, the safety assessment of GMOs involves the identification of the intended and unintended effects as a result of the genetic modification, a.o. through molecular characterisation. For most of the current transgenic crops the molecular characterisation consists of the determination of inserted DNA including the insert sequence, the insertion site(s), the number of inserts and its/their flanking regions. This is currently typically done by Southern blot (SB) and Polymerase chain reaction (PCR) analysis in combination with standard sequencing approaches. Next-generation sequencing (NGS) has had a profound impact on several areas of genomic research. Along with the diverse range of applications of NGS, some recent research projects and product developers explored the use of NGS as a tool for the molecular characterisation of GMOs in view of their risk assessment. JF - 2nd Plant Genomics Congress Europe CP - Global Engage U1 - 493 U2 - 12-05-14 to 13-05-14 ER - TY - JOUR T1 - Comparative study of seven commercial kits for human DNA extraction from urine samples suitable for DNA biomarker-based public health studies. JF - J Biomol Tech Y1 - 2014 A1 - El Bali, Latifa A1 - Diman, Aurélie A1 - Bernard, Alfred A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - Biomarkers KW - Dna KW - Female KW - Genome, Human KW - Genotype KW - Humans KW - Male KW - polymerase chain reaction KW - Polymorphism, Single Nucleotide KW - public health KW - Real-Time Polymerase Chain Reaction KW - Receptors, Nicotinic KW - specimen handling KW - urine AB -

Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at -20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies.

VL - 25 CP - 4 U1 - http://www.ncbi.nlm.nih.gov/pubmed/25365790?dopt=Abstract M3 - 10.7171/jbt.14-2504-002 ER - TY - Generic T1 - The CoSYPS Path Food Project: development of a system for detection and identification of food-borne pathogenic bacteria present in carcass samples according to a "Combinatory qPCR" Technology Y1 - 2014 A1 - E. Barbau-Piednoir A1 - J. Mahillon A1 - Sarah Denayer A1 - N Botteldoorn A1 - Nancy Roosens KW - bacteria KW - CoSYPS KW - detection KW - Development KW - food KW - identification KW - pathogenic KW - present KW - Project KW - report KW - Sample KW - Samples KW - System KW - technology PB - WIV-ISP CY - Brussels, Belgium U1 - 4861 ER - TY - BOOK T1 - Detection of Listeria spp. and Listeria monocytogenes in food and feed products4849 T2 - Listeria monocytogene s: Food sources, Prevalence and management strategies Y1 - 2014 A1 - E. Barbau-Piednoir A1 - J. Mahillon A1 - Nancy Roosens A1 - N Botteldoorn ED - E. C. Hambrick KW - detection KW - Development KW - feed KW - food KW - food sources KW - Listeria KW - Listeria monocytogenes KW - Listeria spp KW - management KW - prevalence KW - PRODUCTS KW - Research KW - Strategies KW - Strategy JF - Listeria monocytogene s: Food sources, Prevalence and management strategies PB - Nova Science Publishers CY - USA SN - 978-1-63117-054-6 CP - 8 U1 - 4849 M3 - not available ER - TY - Generic T1 - Development of a Luminex®-based molecular alternative for classical microbiological subtyping techniques, Salmonella phage typing as a case study Y1 - 2014 A1 - Wuyts,V. A1 - Sophie Bertrand A1 - Nancy Roosens A1 - Marchal,K. A1 - Sigrid C.J. De Keersmaecker ED - Johan Peeters KW - a KW - accuracy KW - aims KW - alternative KW - an KW - AS KW - Belgium KW - Case KW - case studies KW - Case study KW - case-study KW - cause KW - Common KW - Comparison KW - Confinement KW - detection KW - Development KW - foo KW - food KW - general KW - hum KW - Human KW - identify KW - INFECTION KW - intoxication KW - IS KW - IT KW - lar subtyping KW - Lead KW - Marker KW - Markers KW - method KW - methods KW - Molecular KW - molecular subtyping KW - monella KW - Mul KW - Multiplex assay KW - need KW - needs KW - Objective KW - ole KW - ON KW - outbreak KW - pathogen KW - Phage type KW - plex assay KW - present KW - S KW - Salmonella KW - SCREENING KW - SELECTED KW - SOFTWARE KW - study KW - Subtyping KW - Surveillance KW - Target KW - Technique KW - Type KW - typing AB - The PhD-project SalMolType aims to develop an alternative, molecular method for the subtyping of a pathogen. Subtyping of pathogens is essential for surveillance and timely confinement of an outbreak, since it enables us to identify a link between a human isolate and the source of an infection. Molecular techniques allow, in comparison to traditional microbiological subtyping methods, a more convenient standardisation, higher accuracy, they are faster and, in general, more objective. PB - WIV-ISP CY - Brussels, Belgium SN - D/2014/2505/35 U1 - 39122 ER - TY - Generic T1 - Development of a multiplex liquid bead array-based detection system for genetic characterization of pathogenic E. coli 4869 Y1 - 2014 A1 - E. Barbau-Piednoir A1 - N Botteldoorn A1 - Dierckx,D. A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - a KW - conference KW - detection KW - Development KW - e KW - e.coli KW - food KW - Food Microbiology KW - Genetic KW - Luminex KW - microbiology KW - ON KW - pathogenic KW - STEC KW - System KW - VTEC JF - Nineteenth Conference on Food Microbiology PB - NA CY - NA CP - =Belgian Society for Food Microbiology vzw/aslb (BSFM) U1 - 38504 U2 - 18-19/09/2014 ER - TY - Generic T1 - Evaluation and implementation of molecular tools to use epigenetic biomarkers present in urine for public health research Y1 - 2014 A1 - Balzarini,S. A1 - Diman,A. A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker ED - Gabbianell,R. ED - A.J. Martinez ED - De Caterina,R. KW - Biomarkers KW - European KW - EVALUATION KW - health KW - health research KW - implementation KW - Molecular KW - ON KW - present KW - public KW - public health KW - Public-health KW - Research KW - School KW - urine KW - use JF - European Summer School on Nutrigenomic T3 - J Nutrigenet Nutrigenomics VL - 15 CP - Gabbianell,R., Martínez,A.J., De Caterina,R. U1 - 38489 U2 - 1-5/09/2014 ER - TY - Generic T1 - Evaluation of the diversity of indoor airborne molds to assess their impact on public health Y1 - 2014 A1 - Libert,X. A1 - Bladt,S. A1 - Bureau,F. A1 - Camille Chasseur A1 - Ann Packeu A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - Airborne KW - airborne molds KW - application KW - applications KW - diversity KW - EVALUATION KW - health KW - Impact KW - Indoor KW - Indoor air KW - NGS KW - ON KW - public KW - public health KW - Public-health JF - Applications of Next Generation Sequencing for public health PB - NA CY - NA CP - =WIV-ISP U1 - 4875 U2 - 10/10/2014 ER - TY - Generic T1 - Evaluation of urine as source of DNA biomarkers for studies supporting a pro-active public health policy Y1 - 2014 A1 - L. El Bali A1 - A. Bernard A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - a KW - AS KW - Biomarkers KW - Diagnostics KW - Dna KW - DNA biomarkers KW - EVALUATION KW - health KW - HEALTH POLICIES KW - HEALTH POLICY KW - POLICIES KW - POLICY KW - public KW - public health KW - Public-health KW - study KW - symposium KW - urine KW - urine,Biomarkers,DNA,Public Health JF - 2nd Austrian Biomarker Symposium 2014: Early Diagnostics PB - NA CY - NA CP - Austrian Institute of Technology (AIT) U1 - 4855 U2 - 31/03-01/04/2014 ER - TY - JOUR T1 - Evaluation of viability-qPCR detection system on viable and dead Salmonella serovar Enteritidis. JF - J Microbiol Methods Y1 - 2014 A1 - Barbau-Piednoir, Elodie A1 - Mahillon, Jacques A1 - Pillyser, Julie A1 - Wim Coucke A1 - Nancy Roosens A1 - N Botteldoorn KW - Azides KW - Microbial Viability KW - Propidium KW - Real-Time Polymerase Chain Reaction KW - Salmonella enteritidis KW - Salmonella Infections KW - Sensitivity and Specificity AB -

The propidium monoazide (PMA) coupled with PCR (viability PCR) is used in foodborne pathogen detection in order to detect only viable bacteria. Originally presented to fully remove the signal of dead bacteria, the limits of the viability PCR rapidly came out in the literature. In this study, the use of PMA in a viability-qPCR (v-qPCR) was assessed on viable and dead cells of Salmonella enterica subsp. enterica serovar Enteritidis. The PMA treatment protocol was modified (dark incubation duration, concentration of PMA) to evaluate if a complete negative signal of dead Salmonella was possible. However, none of these modifications was found to improve the removal of the remaining qPCR signal observed in the presence of dead bacteria. The present research also underlines that PMA may unexpectedly decrease the qPCR signal observed on living S. Enteritidis at low concentration. Finally, the use of S. Enteritidis cells killed by processes altering or not the cell-wall/membrane gives us a clue to answering the question about the non-total extinction of the signal of dead cells sample in the v-qPCR assay. Indeed, the data strongly indicate that the remaining qPCR signal observed in non-culturable cells does not only depend on the cell-wall/membrane integrity of the bacteria. According to these results, the authors suggest that for a rapid and reliable foodborne bacteria detection system, an enrichment followed by a qPCR analysis should be preferred to a v-qPCR.

VL - 103 U1 - http://www.ncbi.nlm.nih.gov/pubmed/24927988?dopt=Abstract M3 - 10.1016/j.mimet.2014.06.003 ER - TY - Generic T1 - Evaluation of whole genome sequencing for routine subtyping of Salmonella Typhimurium for surveillance and outbreak investigation Y1 - 2014 A1 - Wuyts,V. A1 - Sophie Bertrand A1 - Wesley Mattheus A1 - Nancy Roosens A1 - Marchal,K. A1 - Sigrid C.J. De Keersmaecker ED - WIV-ISP KW - application KW - applications KW - EVALUATION KW - Genome KW - health KW - investigation KW - NGS KW - outbreak KW - public KW - public health KW - Public-health KW - routine KW - Salmonella KW - Salmonella typhimurium KW - Subtyping KW - Surveillance KW - whole genome KW - whole genome sequencing JF - Applications of Next Generation Sequencing for public health CP - WIV-ISP U1 - 39123 U2 - 10/10/2014 ER - TY - JOUR T1 - Genome Sequence of the Salmonella enterica subsp. enterica Serovar Namur Strain 05-2929, Lacking the Salmonella Atypical Fimbrial Operon. JF - Genome Announc Y1 - 2014 A1 - Barbau-Piednoir, Elodie A1 - Sophie Bertrand A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker AB -

This paper announces the genome sequence and annotation of Salmonella enterica subsp. enterica serovar Namur strain 05-2929. S. Namur is a new serovar (39:z4,z23:-) that was isolated from a patient with salmonellosis in 2005 in Namur, Belgium, and has been identified as lacking the Salmonella atypical fimbrial (saf) operon.

VL - 2 CP - 2 U1 - http://www.ncbi.nlm.nih.gov/pubmed/24723724?dopt=Abstract M3 - 10.1128/genomeA.00299-14 ER - TY - Generic T1 - Genome sequence of the Salmonella enterica subsp. enterica serovar Namur strain 05-2929, lacking the Salmonella atypical fimbrial operon.5090 Y1 - 2014 A1 - E. Barbau-Piednoir A1 - Bertrand,N. A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - application KW - applications KW - Genome KW - health KW - Operon KW - public KW - public health KW - Public-health KW - Salmonella KW - Salmonella enterica KW - serovar Namur KW - shortgun KW - strain KW - whole genome JF - Applications of Next Generation Sequencing for public health PB - NA CY - NA CP - =WIV-ISP U1 - 5090 U2 - 10/10/2014 ER - TY - JOUR T1 - Guidelines for validation of qualitative real-time PCR methods JF - Trends in Food Science & Technology Y1 - 2014 A1 - Sylvia Broeders A1 - Ingrid Huber A1 - L. Grohmann A1 - Berben,G. A1 - Isabel Taverniers A1 - Mazzara,M. A1 - Nancy Roosens A1 - Morisset,D. KW - an KW - approach KW - approaches KW - Area KW - Areas KW - AS KW - detection KW - genetically KW - Genetically modified KW - Genetically modified organism KW - Genetically modified organisms KW - GMO KW - GMO detection KW - GMO screening KW - Guidelines KW - implement KW - IS KW - method KW - methods KW - ON KW - PCR KW - polymerase chain reaction KW - qPCR KW - qualitative KW - real time PCR KW - Real-time PCR KW - Real-Time Polymerase Chain Reaction KW - SCREENING KW - Screening method KW - specific KW - study KW - technology KW - TESTING KW - VALIDATION KW - Validation study AB - As for many areas of molecular testing, detection of Genetically Modified Organisms (GMO) relies on the real-time Polymerase Chain Reaction (qPCR) technology. Due to the increasing number of GMO, a screening approach using qualitative screening methods has become an integrated part of GMO detection. However, specific guidelines for the validation of these methods are lacking. Here, a pragmatic approach to conduct in-house and inter-laboratory validation studies for GMO screening methods, is proposed. Such guidelines could be adapted to other areas where qualitative qPCR methods are used for molecular testing allowing to implement easily a more reliable screening phase where necessary. VL - 37 U1 - 4790 M3 - 10.1016/j.tifs.2014.03.008 ER - TY - JOUR T1 - An innovative and integrated approach based on DNA walking to identify unauthorised GMOs JF - Food Chemistry Y1 - 2014 A1 - Marie-Alice Fraiture A1 - Philippe Herman A1 - Taverniers, Isabel A1 - De Loose, Marc A1 - Deforce, Dieter A1 - Nancy Roosens KW - DNA walking KW - GM rice KW - GMO detection KW - Integrated approach KW - pCAMBIA vector KW - Quantitative real-time PCR KW - Transgene flanking region KW - Unauthorised GMO AB -

In the coming years, the frequency of unauthorised genetically modified organisms (GMOs) being present in the European food and feed chain will increase significantly. Therefore, we have developed a strategy to identify unauthorised GMOs containing a pCAMBIA family vector, frequently present in transgenic plants. This integrated approach is performed in two successive steps on Bt rice grains. First, the potential presence of unauthorised GMOs is assessed by the qPCR SYBR®Green technology targeting the terminator 35S pCAMBIA element. Second, its presence is confirmed via the characterisation of the junction between the transgenic cassette and the rice genome. To this end, a DNA walking strategy is applied using a first reverse primer followed by two semi-nested PCR rounds using primers that are each time nested to the previous reverse primer. This approach allows to rapidly identify the transgene flanking region and can easily be implemented by the enforcement laboratories.

VL - 147 M3 - https://doi.org/10.1016/j.foodchem.2013.09.112 ER - TY - JOUR T1 - Inter-laboratory Testing of GMO Detection by Combinatory SYBR®Green PCR Screening (CoSYPS) JF - Food analytical methods Y1 - 2014 A1 - E. Barbau-Piednoir A1 - Stragier,P. A1 - Nancy Roosens A1 - Mazzara,M. A1 - Savini,C. A1 - Van den Eede,G. A1 - Marc Van den Bulcke KW - combinatory KW - CoSYPS KW - detection KW - GMO KW - GMO detection KW - ON KW - PCR KW - SCREENING AB - available on line VL - 7 U1 - 4617 M3 - 10.1007/s12161-014-9837-3 ER - TY - RPRT T1 - La méthylation de l'ADN dans l'urine: Evaluation d'un procédé de conversion au bisulfite de sodium de l'ADN provenant de l'urine, en vue d'en mesurer son état de méthylation Y1 - 2014 A1 - Phillipot,S. A1 - Diman,A. A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - bisulfite de sodium KW - de KW - EN KW - EVALUATION KW - methylation KW - sodium KW - urine PB - WIV-ISP CY - Brussels, Belgium SN - D/2014/2505/43 U1 - 38858 ER - TY - JOUR T1 - New trait-specific SYBR(R)Green qPCR methods to expand the panel of GMO screening methods used in the CoSYPS JF - Eur Food Res Technol Y1 - 2014 A1 - Sylvia Broeders A1 - Marie-Alice Fraiture A1 - Els Vandermassen A1 - Delvoye,M. A1 - E. Barbau-Piednoir A1 - A. Lievens A1 - Nancy Roosens KW - / KW - CoSYPS KW - GMO KW - GMO screening KW - method KW - methods KW - qPCR KW - SCREENING KW - Screening method KW - SYBR(r)Green AB - / VL - / U1 - 5084 M3 - http://dx.doi.org/10.1007/s00217-015-2454-6 ER - TY - Generic T1 - A new type of multiplex assay to screen for GMO in food and feed samples developed within the ORIENT-EXPRESS project4851 Y1 - 2014 A1 - Sylvia Broeders A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens KW - a KW - abstract KW - feed KW - food KW - GMO KW - Multiplex assay KW - ORIENT-EXPRESS project KW - Project KW - Sample KW - Samples KW - Type AB - No abstract JF - LabInfo VL - 11 U1 - 4851 ER - TY - Generic T1 - NeXSplorer.iph: Development of next generation sequencing data analysis tools in support of a fast response for public health and food chain safety Y1 - 2014 A1 - Sigrid C.J. De Keersmaecker A1 - Sophie Bertrand A1 - Wesley Mattheus A1 - Philippe Herman A1 - Katelijne Dierick A1 - N Botteldoorn A1 - Sarah Denayer A1 - Steven Van Gucht A1 - Isabelle Thomas A1 - Deforce,D. A1 - Marchal,K. A1 - Nancy Roosens KW - a KW - analysi KW - analysis KW - application KW - applications KW - bioinformatics KW - data KW - Development KW - food KW - Food Chain KW - GMO KW - health KW - NGS KW - public KW - public health KW - Public-health JF - Applications of Next Generation Sequencing for public health PB - NA CY - NA CP - =WIV-ISP U1 - 38637 U2 - 10/10/2014 ER - TY - Generic T1 - ORIENT-EXPRESS in times of crisis Y1 - 2014 A1 - Sylvia Broeders A1 - Sigrid C.J. De Keersmaecker A1 - Steven Van Gucht A1 - Isabelle Thomas A1 - Vanessa Suin A1 - Katelijne Dierick A1 - N Botteldoorn A1 - Steven Van Borm A1 - Nancy Roosens KW - application KW - applications KW - arbovirus KW - crisis KW - detection KW - gasto-intestinal parasites KW - GMO KW - health KW - Luminex KW - NGS KW - public KW - public health KW - Public-health KW - qPCR KW - time KW - Times JF - Applications of Next Generation Sequencing for public health PB - NA CY - NA CP - WIV-ISP U1 - 4873 U2 - 10/10/2014 ER - TY - Generic T1 - SYBR Green assay development Y1 - 2014 A1 - Nancy Roosens KW - GM KW - SYBR(r)Green KW - Workshop JF - Interactive workshop for Chinese GM rice detection PB - NA CY - NA CP - LGC U1 - 38872 U2 - 24/06/2014 ER - TY - Generic T1 - Validation of the entire CoSYPS Path Food workflow for qPCR detection, identification, isolation and confirmation of food-borne pathogenic bacteria present in carcass samples Y1 - 2014 A1 - E. Barbau-Piednoir A1 - J. Mahillon A1 - Nancy Roosens KW - bacteria KW - CoSYPS KW - CoSYPS,Salmonella,Listeria,qPCR,entire workflow validation KW - detection KW - Europe KW - food KW - identification KW - method KW - methods KW - pathogenic KW - qPCR KW - Sample KW - Samples KW - VALIDATION JF - Rapid Methods Europe (RME) 2014 PB - NA CY - NA CP - RME advisory committee U1 - 4621 U2 - 31/03/2014-02/04/2014 ER - TY - Generic T1 - Agenda Point 2.4: Progress report of AG SMV Y1 - 2013 A1 - Nancy Roosens KW - AG SMV KW - ENGL KW - meeting KW - progress report KW - report U1 -

38870

ER - TY - RPRT T1 - Annual report of the GMO lab concerning the validation dossiers for identification of Genetically Modified Organisms (GMO) Y1 - 2013 A1 - N. Papazova A1 - Sylvia Broeders A1 - Els Vandermassen A1 - Delvoye,M. A1 - D. Van Geel A1 - M Demarteau A1 - Nancy Roosens A1 - Anne-Marie Vanherle KW - annual report KW - genetically KW - Genetically modified KW - Genetically modified organism KW - Genetically modified organisms KW - GMO KW - identification KW - report KW - VALIDATION PB - WIV-ISP, PBB CY - Brussels, Belgium SN - D/2013/2505/28 U1 - 451 ER - TY - JOUR T1 - Development and validation of qualitative SYBR®Green real-time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes. JF - Appl Microbiol Biotechnol Y1 - 2013 A1 - Barbau-Piednoir, Elodie A1 - N Botteldoorn A1 - Yde, Marc A1 - Mahillon, Jacques A1 - Nancy Roosens KW - Bacterial Proteins KW - Food Microbiology KW - Listeria KW - Listeria monocytogenes KW - Real-Time Polymerase Chain Reaction AB -

A combination of four qualitative SYBR®Green qPCR screening assays targeting two levels of discrimination: Listeria genus (except Listeria grayi) and Listeria monocytogenes, is presented. These assays have been developed to be run simultaneously using the same polymerase chain reaction (PCR) programme. The paper also proposes a new validation procedure to specifically validate qPCR assays applied to food microbiology according to two guidelines: the ISO 22118 norm and the "Definition of minimum performance requirements for analytical methods of GMO testing". The developed assays target the iap, prs and hlyA genes that belong to or neighbour the virulence cluster of Listeria spp. The selected primers were designed to amplify short fragments (60 to 103 bp) in order to obtain optimal PCR efficiency (between 97 and 107 % efficiency). The limit of detection of the SYBR®Green qPCR assays is two to five copies of target genes per qPCR reaction. These assays are highly accurate (98.08 and 100 % accuracy for the Listeria spp. and L. monocytogenes assays, respectively).

VL - 97 CP - 9 U1 - http://www.ncbi.nlm.nih.gov/pubmed/23086339?dopt=Abstract M3 - 10.1007/s00253-012-4477-2 ER - TY - Generic T1 - Development of a HRM-based detection method for DNA biomarkers in support of public health Y1 - 2013 A1 - Sigrid C.J. De Keersmaecker A1 - Delvoye,M. A1 - Ahdach,O. A1 - L. El Bali A1 - Nancy Roosens ED - Pfaffl,S. ED - Pfaffl,M. KW - a KW - Biomarkers JF - qPCR & NGS 2013 Event: Next Generation Thinking in Molecular Diagnostics T3 - Symposium Proceedings of the 6th international qPCR Symposium & Industrial Exhibition & Application Workshops: Next Generation Thinking in Molecular Diagnostics CY - Technical University Munich SN - 9783000410246 CP - Pfaffl,S., Pfaffl,M. U1 - 38635 U2 - 18/03/2013-20/03/2013 ER - TY - Generic T1 - Development of a molecular subtyping method for Salmonella Typhimurium combining different types of markers in a Luminex xTAG assay Y1 - 2013 A1 - Wuyts,V. A1 - Sophie Bertrand A1 - Nancy Roosens A1 - Marchal,K. A1 - Sigrid C.J. De Keersmaecker ED - Luminex KW - 2008 KW - a KW - Antibiotic KW - Antibiotic resistance KW - Antimicrobial KW - Antimicrobial resistance KW - Belgium KW - cause KW - Common KW - concept KW - Confinement KW - Correlation KW - Design KW - detection KW - Development KW - disease KW - Diseases KW - Foodborne Diseases KW - gene KW - Genes KW - Genome KW - identification KW - identify KW - IS KW - Luminex KW - Marker KW - Markers KW - method KW - Molecular KW - molecular subtyping KW - Multiplex assay KW - ON KW - outbreak KW - outbreaks KW - Phage type KW - present KW - profile KW - Profiles KW - resistance KW - S KW - Salmonella KW - Salmonella enterica KW - Salmonella typhimurium KW - SELECTED KW - stability KW - strain KW - Strategies KW - Strategy KW - Subtyping KW - Surveillance KW - Target KW - technology KW - Type AB - Subtyping of JF - xSAMPLES Benelux 2013 CP - Luminex U1 - 438 U2 - 19-20/06/2013 ER - TY - Generic T1 - DNA from urine as source for biomarkers in public health Y1 - 2013 A1 - L. El Bali A1 - A. Bernard A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - Biomarkers KW - Dna KW - public health KW - urine JF - Biomarker discovery:Driving technologies PB - NA CY - NA CP - =Euroscicon U1 - 38706 U2 - 28/02/2013 ER - TY - Generic T1 - GGO in voeding: detectie, identificatie en kwantificatie Y1 - 2013 A1 - Sylvia Broeders A1 - Nancy Roosens KW - GGO KW - voeding PB - WIV-ISP CY - Brussels, Belgium SN - D/2013/2505/22 U1 - 440 ER - TY - Generic T1 - Global status of the GMO cultivation and authorisation Y1 - 2013 A1 - Nancy Roosens KW - Communication KW - global KW - global statuts KW - GMO KW - Group KW - status JF - NRL-GMO Communication group PB - NA CY - NA CP - NRL-GGO/FAVV U1 - 38869 U2 - 06/06/2013 ER - TY - JOUR T1 - The GMOseek matrix: a decision support tool for optimizing the detection of genetically modified plants JF - BMC Bioinformatics Y1 - 2013 A1 - A. Block A1 - Frédéric Debode A1 - L. Grohmann A1 - Hulin,Julie A1 - Isabel Taverniers A1 - L. Kluga A1 - E. Barbau-Piednoir A1 - Sylvia Broeders A1 - Ingrid Huber A1 - Marc Van den Bulcke A1 - Heinze,P. A1 - Berben,G. A1 - U. Busch A1 - Nancy Roosens A1 - Eric Janssen A1 - Jana Zel A1 - Kristina Gruden A1 - Morisset,D. KW - ALL KW - an KW - analysi KW - analysis KW - application KW - applications KW - AS KW - at KW - Commercialization KW - composition KW - data KW - Database KW - Decision KW - detection KW - detection method KW - Development KW - Dna KW - effective KW - genetically KW - Genetically modified KW - genetically modified plant KW - Genetically modified plants KW - Genome KW - GMO KW - GMO detection KW - GMOs KW - GMOseek KW - GMOseek matrix KW - INFORMATION KW - IS KW - matrix KW - method KW - methods KW - occurrence KW - ON KW - ORIGIN KW - plant KW - plant origin KW - Plants KW - PRODUCTS KW - result KW - results KW - SCREENING KW - specific KW - status KW - Strategies KW - Strategy KW - Target KW - Targets KW - Transgene KW - use AB - Background: Since their first commercialization, the diversity of taxa and the genetic composition of transgenesequences in genetically modified plants (GMOs) are constantly increasing. To date, the detection of GMOs andderived products is commonly performed by PCR-based methods targeting specific DNA sequences introducedinto the host genome. Information available regarding the GMOs' molecular characterization is dispersed and notappropriately organized. For this reason, GMO testing is very challenging and requires more complex screeningstrategies and decision making schemes, demanding in return the use of efficient bioinformatics tools relying onreliable information.Description: The GMOseek matrix was built as a comprehensive, online open-access tabulated database whichprovides a reliable, comprehensive and user-friendly overview of 328 GMO events and 247 different geneticelements (status: 18/07/2013). The GMOseek matrix is aiming to facilitate GMO detection from plant origin atdifferent phases of the analysis. It assists in selecting the targets for a screening analysis, interpreting the screeningresults, checking the occurrence of a screening element in a group of selected GMOs, identifying gaps in theavailable pool of GMO detection methods, and designing a decision tree. The GMOseek matrix is an independentdatabase with effective functionalities in a format facilitating transferability to other platforms. Data were collectedfrom all available sources and experimentally tested where detection methods and certified reference materials(CRMs) were available.Conclusions: The GMOseek matrix is currently a unique and very valuable tool with reliable information on GMOsfrom plant origin and their present genetic elements that enables further development of appropriate strategies forGMO detection. It is flexible enough to be further updated with new information and integrated in differentapplications and platforms. VL - 14 CP - 256 U1 - 35191 M3 - http://dx.doi.org/10.1186/1471-2105-14-256 ER - TY - Generic T1 - Harmonised approaches for validation of methods for GMO detection in the NRL according to the last EU guidelines445 Y1 - 2013 A1 - N. Papazova A1 - Nancy Roosens A1 - Isabel Taverniers A1 - M. De Loose KW - / KW - approach KW - approaches KW - detection KW - EU KW - GMO KW - GMO detection KW - Guidelines KW - method KW - methods KW - VALIDATION AB - / JF - LabInfo VL - 10 CP - juil-13 U1 - 38825 ER - TY - Generic T1 - An innovative integrated approach based on DNA walking to identify unauthorized GMOs Y1 - 2013 A1 - Marie-Alice Fraiture A1 - Philippe Herman A1 - Taverniers, Isabel A1 - De Loose, Marc A1 - Nancy Roosens KW - An innovative integrated approach based on DNA walking to identify unauthorized GMOs AB -

In the next coming years, the frequency of unauthorized genetically modified organisms (GMOs) being present in the European food/feed chain will increase significantly. Rice already constitutes a challenge for laboratories developing methods to detect unauthorized GMOs. Indeed, in 2012, several genetic modified rices were detected in products imported from Asia, mainly from China. Therefore, we have developed a strategy to identify unauthorized GMOs containing a pCAMBIA family vector, frequently present in transgenic plants. The presented integrated approach is performed in two main successive steps on Bt rice grains. First, the potential presence of unauthorized GMOs is assessed by the qPCR SYBR®Green technology targeting the terminator 35S (t35S) pCAMBIA element, which allows discriminating pCAMBIA family vectors. Second, its presence is confirmed via the characterization of the junction between the transgenic cassette and the rice genome. To this end, a DNA walking strategy is applied using a first reverse primer followed by two semi-nested PCR rounds using primers that are each time nested to the previous reverse primer. The sensitivity of the method was assessed. This innovative approach allows to rapidly identifying the transgene flanking region and presents the advantage to be easily implementable in GMO routine analysis by the enforcement laboratories.

PB - Trends in Food Analysis VII Symposium CY - Gent, Belgium ER - TY - JOUR T1 - MLVA as a tool for public health surveillance of human Salmonella Typhimurium: prospective study in Belgium and evaluation of MLVA loci stability. JF - PLoS One Y1 - 2013 A1 - Wuyts, Véronique A1 - Wesley Mattheus A1 - Guillaume De Laminne de Bex A1 - Wildemauwe, Christa A1 - Nancy Roosens A1 - Marchal, Kathleen A1 - Sigrid C.J. De Keersmaecker A1 - Sophie Bertrand KW - Bacterial Typing Techniques KW - Belgium KW - Disease Outbreaks KW - Humans KW - Minisatellite Repeats KW - Prospective Studies KW - Public Health Surveillance KW - Salmonella Infections KW - Salmonella typhimurium AB -

Surveillance of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) is generally considered to benefit from molecular techniques like multiple-locus variable-number of tandem repeats analysis (MLVA), which allow earlier detection and confinement of outbreaks. Here, a surveillance study, including phage typing, antimicrobial susceptibility testing and the in Europe most commonly used 5-loci MLVA on 1,420 S. Typhimurium isolates collected between 2010 and 2012 in Belgium, was used to evaluate the added value of MLVA for public health surveillance. Phage types DT193, DT195, DT120, DT104, DT12 and U302 dominate the Belgian S. Typhimurium population. A combined resistance to ampicillin, streptomycin, sulphonamides and tetracycline (ASSuT) with or without additional resistances was observed for 42.5% of the isolates. 414 different MLVA profiles were detected, of which 14 frequent profiles included 44.4% of the S. Typhimurium population. During a serial passage experiment on selected isolates to investigate the in vitro stability of the 5 MLVA loci, variations over time were observed for loci STTR6, STTR10, STTR5 and STTR9. This study demonstrates that MLVA improves public health surveillance of S. Typhimurium. However, the 5-loci MLVA should be complemented with other subtyping methods for investigation of possible outbreaks with frequent MLVA profiles. Also, variability in these MLVA loci should be taken into account when investigating extended outbreaks and studying dynamics over longer periods.

VL - 8 CP - 12 U1 - http://www.ncbi.nlm.nih.gov/pubmed/24391880?dopt=Abstract M3 - 10.1371/journal.pone.0084055 ER - TY - Generic T1 - Molecular subtyping of Salmonella Typhimurium combining different types of markers in a multiplex liquid bead suspension array Y1 - 2013 A1 - Wuyts,V. A1 - Sophie Bertrand A1 - Nancy Roosens A1 - Marchal,K. A1 - Sigrid C.J. De Keersmaecker KW - 2008 KW - Antibiotic KW - Antimicrobial KW - Antimicrobial resistance KW - Belgium KW - cause KW - Common KW - Confinement KW - Design KW - detection KW - disease KW - Diseases KW - Foodborne Diseases KW - gene KW - Genes KW - Genome KW - identification KW - identify KW - International KW - IS KW - Luminex KW - method KW - molecular subtyping KW - Multiplex assay KW - ON KW - outbreak KW - Phage type KW - profile KW - resistance KW - S KW - Salmonella KW - Salmonella enterica KW - Salmonella typhimurium KW - stability KW - strain KW - Subtyping KW - Surveillance KW - symposium KW - Target KW - Type AB -

Subtyping of

JF - I3S: International Symposium Salmonella and Salmonellosis 2013 PB - NA CY - NA CP - ANSES,InVS,INRA,Institut Pasteur,ISPAIA and ZOOPOLE U1 - 35206 U2 - 27-29/05/2013 ER - TY - JOUR T1 - New SYBR®Green methods targeting promoter sequences used for screening of several GM events pending for authorisation in Europe JF - Eur.Food.Res.Technol. Y1 - 2013 A1 - Sylvia Broeders A1 - E. Barbau-Piednoir A1 - Els Vandermassen A1 - Frédéric Debode A1 - Mazzara,M. A1 - Nancy Roosens KW - an KW - AS KW - consumer KW - Control KW - Cost KW - CoSYPS KW - crops KW - Decision KW - detection KW - effective KW - Europe KW - feed KW - food KW - Genetically modified KW - GM KW - health KW - Institute KW - IS KW - Laboratories KW - matrix KW - method KW - methods AB - Seen the growing number of genetically modified (GM) crops being developed, the need for cost- and time-effective detection methods is increasing to enable continuing the necessary effective control on food and feed products. This need can be achieved by performing an intensive screening combined with decision support tools like the CoSYPS matrix which permits reducing the number of events to be identified. To allow an extra covering power of the CoSYPS and to be able to include new EU-authorised GM events, two new SYBR®Green real-time PCR (qPCR) methods targeting two promoter sequences (pNOS and pFMV) were developed. These methods were validated using acceptance parameters such as the specificity, sensitivity and repeatability. In addition, the methods were transferred to a second laboratory, namely the Institute for Health and Consumer Protection, to test the reproducibility. Furthermore, the applicability and practicability of the methods were tested by using proficiency test samples. The two methods allow a specific and sensitive detection of the targets in food and feed samples and can be used efficiently in different laboratories. VL - 236 U1 - 33874 M3 - http://dx.doi.org/10.1007/s00217-013-1910-4 ER - TY - Generic T1 - New techniques and methods developed by the Belgian NRL-GMO to identify unauthorized GMOs in the UGMMONITOR project Y1 - 2013 A1 - Marie-Alice Fraiture A1 - Philippe Herman A1 - Berben,G. A1 - Frédéric Debode A1 - Eric Janssen A1 - Isabel Taverniers A1 - M. De Loose A1 - Nancy Roosens KW - Belgian KW - GMO KW - GMOs KW - identify KW - method KW - methods KW - New techniques KW - Technique AB - Not available JF - LabInfo VL - 9 U1 - 433 ER - TY - Generic T1 - A new type of multiplex assay to screen for GMO in food and feed samples developed within the ORIENT-EXPRESS project Y1 - 2013 A1 - Sylvia Broeders A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens KW - feed KW - food KW - GMO KW - MO KW - Multiplex assay KW - ORIENT-EXPRESS project KW - Sample KW - Samples KW - Type AB - No abstract JF - LabInfo VL - 11 U1 - 4442 ER - TY - Generic T1 - Nieuwe technieken en methoden ontwikkeld door het Belgische NRL-GGO voor het identificeren van niet-geautoriseerde GGO's in het UGMMONITOR project Y1 - 2013 A1 - Marie-Alice Fraiture A1 - Philippe Herman A1 - Berben,G. A1 - Frédéric Debode A1 - Eric Janssen A1 - Isabel Taverniers A1 - M. De Loose A1 - Nancy Roosens KW - 2012 KW - Belgische KW - biosécurité KW - Database KW - de KW - detection KW - GMO KW - IS KW - OGM KW - santé publique KW - SBB KW - Technique KW - UGM KW - website AB - Het project "UGMMONITOR" bestaat in de ontwikkeling van een platform in de moleculaire biologie en eendatabase (bioveiligheidsinformatie) voor de detectie van UGMs in levensmiddelen en diervoeders. Het wordtgefinancierd door de Federale Overheidsdienst Volksgezondheid, Veiligheid van de Voedselketen en Leefmilieu(overeenkomst RF 11/6242) (begindatum: 1 mei 2012) en zal toelaten om high-tech methoden te ontwikkelenvoor een betere controle op aanwezigheid van UGMs. Tot de doelen van dit project behoren het beschikbaarstellen van een database van UGMs met informatie die nuttig is voor risico-evaluatie, en het ontwikkelen van eengeintegreerde aanpak van high-tech methoden voor detectie van UGMs in levensmiddelen/diervoeders. JF - LabInfo VL - 9 CP - Janvier 2013 U1 - 4050 ER - TY - Generic T1 - Nouvelles techniques et méthodes développées par le LNR-OGM Belge pour identifier les OGMs non-autorisés dans le projet UGMMONITOR Y1 - 2013 A1 - Marie-Alice Fraiture A1 - Philippe Herman A1 - Berben,G. A1 - Frédéric Debode A1 - Eric Janssen A1 - Isabel Taverniers A1 - M. De Loose A1 - Nancy Roosens KW - 2012 KW - Belge KW - biosécurité KW - de KW - detection KW - GMO KW - INFORMATION KW - LE KW - OGM KW - PAR KW - relatives KW - risques KW - santé KW - santé publique KW - SBB KW - Service KW - Technique KW - UGM KW - website AB - Le projet intitule "UGMMONITOR" consiste à développer une plateforme de biologie moleculaire et une base dedonnées (informations relatives à la biosécurité) pour la détection d'UGMs dans la chaîne alimentaire humaine etanimale. Ce projet est financé par le Service Publique Fédéral Santé Publique, Sécurité de la Chaîne Alimentaire etEnvironnement (convention RF 11/6242) (date de début : 01 mai 2012) et permettra de développer des methodeshigh-tech afin d'assurer une meilleure gestion du contrôle des UGMs. Un des principaux objectifs de ce projet estde fournir une base de données d'UGMs contenant des informations utiles pour l'évaluation des risques et aussiune approche high-tech intégrée pour détecter les UGMs dans la chaîne alimentaire humaine et animale. JF - LabInfo VL - 9 CP - Janvier 2013 U1 - 4049 ER - TY - Generic T1 - Plateforme biomoléculaire High Tech et laboratoire d'orientation (orientation rapide de pathogène d'origine inconnue) Y1 - 2013 A1 - Nancy Roosens KW - de KW - ET KW - paris KW - pathogène JF - Réunion WIV-ISP et Réseau Pasteur (Paris, Lille) PB - NA CY - NA CP - WIV-ISP U1 - 4847 U2 - 05/12/2013 ER - TY - Generic T1 - Progress Report of the ENGL Advisory group for Method validation Y1 - 2013 A1 - Nancy Roosens KW - ENGL KW - Group KW - meeting KW - method KW - Method validation KW - progress report KW - report KW - VALIDATION JF - 19th ENGL Plenary meeting PB - NA CY - NA CP - JRC,IHCP U1 - 38868 U2 - 20/06/2013 ER - TY - Generic T1 - Projet UGMMONITOR : résultat de la première année. Y1 - 2013 A1 - Marie-Alice Fraiture A1 - Philippe Herman A1 - Nancy Roosens KW - de KW - UGMMONITOR JF - Premier comité d'accompagnement du projet UGMMONITOR PB - NA CY - NA CP - WIV-ISP,PBB U1 - 38721 U2 - 22/04/2013 ER - TY - Generic T1 - SYBR ® Green qPCR Salmonella detection system allowing discrimination at the genus, species and subspecies levels Y1 - 2013 A1 - E. Barbau-Piednoir A1 - Nancy Roosens A1 - Sophie Bertrand A1 - J. Mahillon A1 - N Botteldoorn ED - BSFM KW - at KW - conference KW - detection KW - discrimination KW - food KW - Food Microbiology KW - LEVEL KW - levels KW - microbiology KW - ON KW - qPCR KW - Salmonella KW - Species KW - System JF - 18th conference on food microbiology T3 - Eighteenth conference on food microbiology CP - Belgian Society of Food Microbiology U1 - 4845 U2 - 12-13/09/2013 ER - TY - JOUR T1 - SYBR®Green qPCR Salmonella detection system allowing discrimination at the genus, species and subspecies levels. JF - Appl Microbiol Biotechnol Y1 - 2013 A1 - Barbau-Piednoir, Elodie A1 - Sophie Bertrand A1 - Mahillon, Jacques A1 - Nancy Roosens A1 - N Botteldoorn KW - Bacterial Proteins KW - Bacteriological Techniques KW - DNA Primers KW - Fluorescent Dyes KW - Organic Chemicals KW - Real-Time Polymerase Chain Reaction KW - Reproducibility of Results KW - Salmonella KW - Sensitivity and Specificity KW - Staining and Labeling AB -

In this work, a three-level Salmonella detection system based on a combination of seven SYBR®Green qPCR was developed. This detection system discriminates Salmonella at the genus, species and subspecies levels using a single 96-well plate. The SYBR®Green qPCR assays target the invA, rpoD, iroB and safC genes, as well as the STM0296 locus, putatively coding for a cytoplasmic protein. This study includes the design of primer pairs, in silico and in situ selectivity, sensitivity, repeatability and reproducibility evaluations of the seven SYBR®Green qPCR assays. Each detection level displayed a selectivity of 100 %. This combinatory SYBR®Green qPCR system was also compared with three commercially available Salmonella qPCR detection kits. This comparison highlighted the importance of using a multi-gene detection system to be able to detect every target strain, even those with deletion or mutation of important genes.

VL - 97 CP - 22 U1 - http://www.ncbi.nlm.nih.gov/pubmed/24113820?dopt=Abstract M3 - 10.1007/s00253-013-5234-x ER - TY - Generic T1 - Validation of qualitative qPCR methods Y1 - 2013 A1 - Sylvia Broeders A1 - Els Vandermassen A1 - Nancy Roosens KW - analysi KW - analysis KW - food KW - Food Analysis KW - method KW - methods KW - qPCR KW - qualitative KW - trend KW - trends KW - VALIDATION JF - Trends in Food Analysis VII T3 - Trends in Food Analysis VII PB - NA CY - KVCV - Sectie Voeding CP - KVCV - Sectie Voeding U1 - 455 U2 - 19/09/2013 M3 - A4044.0211 ER - TY - RPRT T1 - Advanced Analysis of Real Time PCR data Y1 - 2012 A1 - A. Lievens A1 - S. Van Aelst A1 - Marc Van den Bulcke A1 - Goetghebeur,E. A1 - Nancy Roosens KW - analysi KW - analysis KW - data KW - PCR KW - real time PCR KW - time PB - WIV-ISP CY - Brussels, Belgium VL - 2010-2011 SN - ISSN D/2012/2505/19 U1 - 38801 ER - TY - Generic T1 - Chapter 13 : Why « combinatory qPCR » technology is a useful tool to deal with the detection of "unexepected" GMO. Y1 - 2012 A1 - Sylvia Broeders A1 - Nancy Roosens KW - a KW - combinatory KW - detection KW - GMO KW - IS KW - qPCR KW - symposium KW - technology JF - 6th Stevia symposium T3 - Proceeding of the 6th Stevia symposium organized by EUSTAS 2012 PB - NA CY - NA SN - 978 - 90 - 74253-208 CP - EUSTAS 2012 U1 - 417 U2 - 03-04/07/2012 ER - TY - RPRT T1 - Comparative study of five commercial kits for DNA extraction from urine samples. Y1 - 2012 A1 - L. El Bali A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - Comparative Study KW - Dna KW - DNA extraction kits KW - Sample KW - Samples KW - study KW - urine PB - WIV-ISP CY - Brussels SN - D/2012/2505/69 U1 - 442 ER - TY - Generic T1 - Design of multiplex assays for molecular subtyping of pathogens with the Luminex xMAP technology Y1 - 2012 A1 - Wuyts,V. A1 - Nancy Roosens A1 - Sophie Bertrand A1 - Marchal,K. A1 - Sigrid C.J. De Keersmaecker KW - a KW - Advantages KW - Analyte KW - challenge KW - CHALLENGES KW - containment KW - Cost KW - Design KW - detection KW - Development KW - Diagnosis KW - identification KW - IS KW - IT KW - limitation KW - Limitations KW - Luminex KW - microorganism KW - microorganisms KW - Molecular KW - molecular subtyping KW - Multiplex assay KW - Oligonucleotides KW - outbreak KW - outbreaks KW - pathogen KW - reducing KW - regulation KW - Sample KW - SOFTWARE KW - specificity KW - structure KW - Subtyping KW - Surveillance KW - technology KW - Temperature KW - time KW - Type AB -

Multiplex assays are a powerful tool for molecular subtyping of pathogens, which is crucial for rapid diagnosis, surveillance and identification and containment of outbreaks. The Luminex xMAP technology allows detection of up to 500 different analytes per sample in a high-throughput format through a liquid bead suspension array. Different types of Luminex assays can be envisaged. Each assay has its advantages and limitations, some of which are related to the inherent design of the primers and probes used. Software that predicts melting temperatures, hybridization structures and specificity for different types of oligonucleotides, and thereby simulating the multiplex assays in silico can facilitate the design of multiplex assays by reducing time and cost of experimentation.We have compared two commercial software packages for the development and in silico simulation of a ligation dependent amplification (LDA) assay and a direct hybridization assay for the subtyping of a pathogen. The main findings, limitations and challenges will be discussed.

JF - Posttranscriptional regulation and epigenetics in microorganisms PB - NA CY - NA CP - Belgian Society for Microbiology U1 - 428 U2 - 30/11/2012 ER - TY - Generic T1 - Development and full validation of Listeria spp. and Listeria monocytogenes qPCR screening assays to be used in a combinatory system to detect foodborne pathogens Y1 - 2012 A1 - E. Barbau-Piednoir A1 - J. Mahillon A1 - Nancy Roosens A1 - N Botteldoorn KW - a KW - combinatory KW - conference KW - Development KW - food KW - Food Microbiology KW - Listeria KW - Listeria monocytogenes KW - Listeria spp KW - microbiology KW - ON KW - qPCR KW - SCREENING KW - System KW - VALIDATION JF - 17th Conference on Food Microbiology T3 - Seventeenth Conference on Food Microbiology PB - NA CY - NA CP - Belgian Society for Food Microbiology U1 - 422 U2 - Belgian Society of Food Microbiology ER - TY - Generic T1 - Development and full validation of Listeria spp. and Listeria monocytogenes qPCR screening assays to be used in a combinatory system to detect foodborne pathogens Y1 - 2012 A1 - E. Barbau-Piednoir A1 - J. Mahillon A1 - Nancy Roosens A1 - N Botteldoorn ED - International Committee Hygiene KW - 2012 KW - a KW - combinatory KW - conference KW - Development KW - food KW - Food Microbiology KW - global KW - Listeria KW - Listeria monocytogenes KW - Listeria spp KW - microbiology KW - ON KW - qPCR KW - SCREENING KW - System KW - VALIDATION JF - Food Micro 2012 : Global Issues in Food Microbiology T3 - Food Micro 2012 : Global Issues in Food Microbiology: Abstract Book CP - International Committee on Food Microbiology and Hygiene U1 - 38500 U2 - 03-06/09/12 ER - TY - BOOK T1 - Development of a molecular platform for GMO detection in food and feed on the basis of "combinatory qPCR" technology4843 T2 - Polymerase Chain Reaction Y1 - 2012 A1 - Sylvia Broeders A1 - N. Papazova A1 - Marc Van den Bulcke A1 - Nancy Roosens ED - Patricia Hernandez-Gomez KW - a KW - detection KW - Development KW - feed KW - food KW - GMO KW - GMO detection KW - Molecular KW - ON KW - polymerase chain reaction KW - technology JF - Polymerase Chain Reaction PB - =InTech CY - Janeza Trdine 9, 51000 Rijeka, Croatia SN - ISBN 978-953-51-0612-8 CP - 18 U1 - 4843 M3 - DOI: 10.5772/37898 ER - TY - Generic T1 - Emerging threats: new GMO and UGM419 Y1 - 2012 A1 - Sylvia Broeders A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens KW - abstract KW - GMO KW - UGM AB - no abstract JF - LabInfo VL - 8 U1 - 38538 ER - TY - JOUR T1 - Four new SYBR®Green qPCR screening methods for the detection of Roundup Ready(r), LibertyLink(r), and CryIAb traits in genetically modified products362 JF - Eur Food Res Technol Y1 - 2012 A1 - E. Barbau-Piednoir A1 - A. Lievens A1 - Els Vandermassen A1 - G.E. Mbongolo-Mbella A1 - Amaya Leunda A1 - Nancy Roosens A1 - Myriam Sneyers A1 - Marc Van den Bulcke KW - a KW - ALL KW - an KW - analysi KW - analysis KW - application KW - AS KW - at KW - Bacillus KW - Bacillus thuringiensis KW - Cost KW - COSTS KW - CoSYPS KW - detection KW - Efficiency KW - Feed/food analysis KW - genetically KW - Genetically modified KW - Genetically modified organism KW - Genetically modified organisms KW - global KW - GM KW - GMO KW - GMO detection KW - Herbicide resistance KW - identification KW - Insect resistance KW - IS KW - LEVEL KW - levels KW - method KW - methods KW - PCR KW - present KW - PRODUCTS KW - qPCR KW - Quantitative real-time PCR KW - r KW - RANGE KW - result KW - results KW - Sample KW - Samples KW - SCREENING KW - Screening method KW - SENSITIVITY KW - specific KW - specificity KW - SYBR(r)Green KW - SYBRgreen KW - Target KW - Targets KW - Test KW - Traits AB - SYBR(r)Green qPCR methods for the detection of the Roundup Ready (r) "CP4-EPSPS", LibertyLink (r) "PAT" and "BAR" and the Bacillus thuringiensis "CryIAb" traits as present in genetically modified organisms (GMO) were developed. Their specificity, sensitivity, and PCR method efficiency were determined. All methods are specific and generate amplicons of 108, 73, 109, and 69 bp, respectively, for "CP4-EPSPS", "CryIAb", "PAT" and "BAR" targets. They perform well at low target levels and can detect down to 5 copies of their respective targets extracted from a sample. The PCR efficiency of the methods ranges between 91 and 109%. Due to their trait-specific nature, these methods allow an efficient screening between the different GMO. In this way, the number of possible GMO candidates present in a sample can be reduced what results in lower global costs due to limiting of further the number of analytical identification steps. The application of these methods in CoSYPS GMO analysis is illustrated using two GEMMA proficiency test samples and a reference material from the GM rapeseed event RF3. This set of SYBR(r)Green qPCR trait-specific methods represents a very interesting novel set of GMO analysis methods allowing cost-effective identification of GM materials in products. VL - 234 SN - 1438-2377 CP - 1 U1 - 38507 M3 - DOI: 10.1007/s00217-011-1605-7 ER - TY - JOUR T1 - How to Deal with the Upcoming Challenges in GMO Detection in Food and Feed424 JF - Journal of Biomedicine and Biotechnology Y1 - 2012 A1 - Sylvia Broeders A1 - Sigrid C.J. De Keersmaecker A1 - Nancy Roosens KW - a KW - Agriculture KW - ALL KW - an KW - approach KW - approaches KW - challenge KW - CHALLENGES KW - CODING KW - continue KW - Countries KW - crops KW - detection KW - detection method KW - Evolution KW - feed KW - food KW - future KW - Genetic KW - Genome KW - GM KW - GMO KW - GMO detection KW - history KW - Increase KW - IS KW - Laboratories KW - legislation KW - method KW - methods KW - need KW - PCR KW - plant KW - real time PCR KW - Real-time PCR KW - Sample KW - SCREENING KW - technology KW - traceability KW - Transgenic KW - Type AB - Biotech crops are the fastest adopted crop technology in the history of modern agriculture. The commercialisation of GMO is in many countries strictly regulated laying down the need for traceability and labelling. To comply with these legislations, detection methods are needed. To date, GM events have been developed by the introduction of a transgenic insert (i.e., promoter, coding sequence, terminator) into the plant genome and real-time PCR is the detection method of choice. However, new types of genetic elements will be used to construct new GMO and new crops will be transformed. Additionally, the presence of unauthorised GMO in food and feed samplesmight increase in the near future. To enable enforcement laboratories to continue detecting all GMevents and to obtain an idea of the possible presence of unauthorised GMO in a food and feed sample, an intensive screening will become necessary. A pragmatic, cost-e VL - 2012 U1 - 38539 M3 - 10.1155/2012/402418 ER - TY - Generic T1 - NRL-GMO: GMOSeek research project (2009 - 2011) for GMO detection361 Y1 - 2012 A1 - Sylvia Broeders A1 - E. Barbau-Piednoir A1 - G.E. Mbongolo-Mbella A1 - N. Papazova A1 - Pouppez,A. A1 - Nancy Roosens A1 - Isabel Taverniers A1 - M. De Loose A1 - Frédéric Debode A1 - Berben,G. A1 - Eric Janssen KW - 2009 KW - abstract KW - detection KW - GMO KW - GMO detection KW - GMOseek KW - Project KW - Research AB - no abstract JF - LabInfo VL - January 2012 U1 - 38537 ER - TY - RPRT T1 - Overview of Methods for DNA and RNA extraction from urine samples Y1 - 2012 A1 - L. El Bali A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker KW - Dna KW - method KW - methods PB - WIV-ISP, PBB CY - Brussels, Belgium SN - D/2012/2505/65 U1 - 38704 ER - TY - RPRT T1 - Rapport annuel du GMOLAB concernant les dossiers de validation pour l'identification des organismes génétiquement modifiés Y1 - 2012 A1 - N. Papazova A1 - Sylvia Broeders A1 - Els Vandermassen A1 - D. Van Geel A1 - De Pril,S. A1 - M Demarteau A1 - Nancy Roosens A1 - Sciacqua,M. A1 - Anne-Marie Vanherle KW - de KW - GMOlab KW - LE KW - rapport KW - rapport annuel KW - VALIDATION PB - WIV-ISP, PBB CY - Brussels, Belgium U1 - 426 ER - TY - Generic T1 - WIV-ISP Scientific report 2010-2011: Practical issues related to the implementation of the recently adopted EU regulation on "Low Level Presence" of GMOs Y1 - 2012 A1 - N. Papazova A1 - De Pril,S. A1 - M Demarteau A1 - Els Vandermassen A1 - D. Van Geel A1 - Sylvia Broeders A1 - Nancy Roosens KW - EU KW - GMO KW - GMOs KW - implementation KW - LEVEL KW - ON KW - p KW - regulation KW - report KW - WIV-ISP PB - WIV-ISP, PBB CY - Brussels, Belgium U1 - 425 ER - TY - Generic T1 - A combinatory qPCR screening system to detect food-borne pathogens. Y1 - 2011 A1 - E. Barbau-Piednoir A1 - J. Mahillon A1 - Nancy Roosens A1 - N Botteldoorn KW - a KW - combinatory KW - food KW - Food Microbiology KW - foodborne pathogenes KW - meeting KW - microbiology KW - pathogen KW - qPCR KW - SCREENING KW - SYBR(r)Green KW - System JF - Sfam summer meeting 2011: Food Microbiology T3 - Sfam summer meeting 2011: Conference Program PB - NA CY - NA CP - The Society for Applied Microbiology (SfAM) U1 - 38498 U2 - 04-07/07/2011 ER - TY - Generic T1 - A combinatory qPCR screening system to detect food-borne pathogens. Y1 - 2011 A1 - E. Barbau-Piednoir A1 - J. Mahillon A1 - Nancy Roosens A1 - N Botteldoorn KW - a KW - combinatory KW - conference KW - Development KW - food KW - Food Microbiology KW - foodborne pathogenes KW - method KW - methods KW - microbiology KW - ON KW - pathogen KW - qPCR KW - SCREENING KW - SYBR(r)Green KW - System JF - 16th Conference on Food Microbiology T3 - Proceedings of the Sixteenth Conference on Food Microbiology PB - NA CY - NA CP - Belgian Society for Food Microbiology U1 - 38496 U2 - 22-23/09/2011 ER - TY - Generic T1 - Development of qPCR screening methods to be used in a combinatory system to detect food-borne pathogens Y1 - 2011 A1 - E. Barbau-Piednoir A1 - J. Mahillon A1 - Nancy Roosens A1 - N Botteldoorn KW - a KW - challenge KW - CHALLENGES KW - combinatory KW - Development KW - foodborne pathogenes KW - global KW - Global Health KW - health KW - International KW - meeting KW - method KW - methods KW - Network KW - pathogen KW - qPCR KW - SCREENING KW - Screening method KW - SYBR(r)Green KW - System KW - young JF - Scientific Meeting of the Young researchers of Institut Pasteur and Institut Pasteur International Network: Challenges in Global Health: Opportunities for Institut Pasteur International Network T3 - Scientific Meeting of the Young researchers of Institut Pasteur and Institut Pasteur International Network: Challenges in Global Health: Opportunities for Institut Pasteur International Network PB - NA CY - NA CP - Réseau International des Institut Pasteur U1 - 38497 U2 - 10/11/2012 ER - TY - Generic T1 - Les techniques de PCR pour le diagnostic rapide des pathogènes dans l'alimentation346 Y1 - 2011 A1 - E. Barbau-Piednoir A1 - Sarah Denayer A1 - J. Mahillon A1 - Nancy Roosens A1 - N Botteldoorn KW - abstract KW - de KW - foodborne pathogenes KW - LE KW - pathogène KW - PCR KW - Technique AB - no abstract JF - Medi-sphère VL - 283 U1 - 38493 ER - TY - RPRT T1 - Rapport annuel 2010-2011 du GMOlab concernant les dossiers de validation pour l'identification des organismes génétiquement modifiés Y1 - 2011 A1 - N. Papazova A1 - Sylvia Broeders A1 - G.E. Mbongolo-Mbella A1 - Els Vandermassen A1 - De Mets,F. A1 - Nancy Roosens A1 - Sciacqua,M. A1 - Anne-Marie Vanherle A1 - Draguet,V. KW - de KW - GMOlab KW - LE KW - OGM identification KW - qPCR KW - rapport KW - rapport annuel KW - TaqMan(r) KW - VALIDATION PB - WIV-ISP; PBB CY - Brussels, Belgium SN - D/2011/2505/32 U1 - 38824 ER - TY - RPRT T1 - Rapport annuel du GMOlab concernant les dossiers de validation pour la quantification des organismes génétiquement modifiés. Y1 - 2011 A1 - N. Papazova A1 - Sylvia Broeders A1 - Els Vandermassen A1 - Nancy Roosens A1 - Sciacqua,M. A1 - Anne-Marie Vanherle KW - analysis KW - de KW - feed KW - food KW - GMO KW - GMOlab KW - LE KW - qPCR KW - Quantification KW - rapport KW - rapport annuel KW - VALIDATION PB - WIV-ISP; PBB CY - Brussels, Belgium SN - ISSN D/2011/2505/33 U1 - 38823 ER - TY - Generic T1 - SAFEFOODERA: Nouvelles méthodes de detection des OGM Y1 - 2011 A1 - Sylvia Broeders A1 - E. Barbau-Piednoir A1 - G.E. Mbongolo-Mbella A1 - N. Papazova A1 - Pouppez,A. A1 - Nancy Roosens KW - de KW - detection KW - GMO KW - OGM KW - qPCR KW - SYBR(r)Green KW - WIV-ISP PB - WIV-ISP CY - Brussels, Belgium SN - (NL) D/2011/2505/12 and (FR) D/2011/2505/13 U1 - 38536 ER - TY - JOUR T1 - SYBR®Green qPCR methods for detection of endogenous reference genes in commodity crops: a step ahead in combinatory screening of Genetically Modified Crops in food and feed products JF - Eur.Food Res.Technol. Y1 - 2011 A1 - G.E. Mbongolo-Mbella A1 - A. Lievens A1 - E. Barbau-Piednoir A1 - Myriam Sneyers A1 - Amaya Leunda A1 - Nancy Roosens A1 - Marc Van den Bulcke KW - an KW - approach KW - approaches KW - at KW - Biomedical and Life Sciences KW - criteria KW - crops KW - detection KW - Development KW - Efficiency KW - EU KW - European KW - European Union KW - feed KW - food KW - gene KW - Genes KW - Genetically modified KW - Genetically modified crop KW - genetically modified crops KW - Genetically modified organism KW - Genetically modified organisms KW - GMO KW - IS KW - Laboratories KW - maize KW - method KW - methods KW - Network KW - Oilseed rape KW - Order KW - PCR KW - performance KW - polymerase chain reaction KW - PRODUCTS KW - qPCR KW - SCREENING KW - Soybean KW - Strategies KW - Strategy KW - sugar AB - Identification of crops present in food and/or feed matrices represents an important step in the screening strategies targeting genetically modified organisms (GMO). Soybean, maize, oilseed rape, rice, cotton, sugar beet and potato are to date the most important sources of genetically modified materials imported in the European Union (EU). In order to allow detection of their presence in an integrated screening approach, a set of SYBR-«Green real-time polymerase chain reaction (qPCR) methods has been developed which can be used under the same assay conditions and at similar efficiency for each of the abovementioned crops. Each qPCR method is shown to meet the performance criteria (i.e. specificity, limit of detection and PCR efficiency) set by the European Network of GMO Laboratories (ENGL). When combined with the equivalent qPCR methods targeting GMO elements, these crop-specific SYBR-«Green qPCR methods can aid the development of an efficient tool for determining GMO presence in food and/or feed products VL - 232 SN - 14382377 CP - 3 U1 - 345 M3 - http://dx.doi.org/10.1007/s00217-010-1408-2 ER - TY - Generic T1 - Validation and measurement uncertainty in GMO analysis Y1 - 2011 A1 - Sylvia Broeders A1 - Nancy Roosens KW - analysi KW - analysis KW - Development KW - GMO KW - measurement KW - measurement uncertainty (MU) KW - uncertainty KW - VALIDATION KW - Workshop JF - EURACHEM workshop 'Recent developments in measurement uncertainty' PB - NA CY - NA CP - =Eurachem U1 - 38535 U2 - 06-07/06/2011 ER - TY - Generic T1 - A combinatory qPCR screening system to detect food-borne pathogens Y1 - 2010 A1 - E. Barbau-Piednoir A1 - J. Mahillon A1 - Nancy Roosens A1 - N Botteldoorn KW - a KW - combinatory KW - conference KW - food KW - Food Microbiology KW - microbiology KW - ON KW - pathogen KW - qPCR KW - SCREENING KW - System JF - 15th Conference on Food Microbiology PB - NA CY - NA CP - Department of Food Safety and Food Quality,UGent U1 - 38492 U2 - 16/09/2010 to 17/09/2010 ER - TY - RPRT T1 - Development of a molecular detection platform for GMO detection in food based on "combinatory qPCR" technology Y1 - 2010 A1 - Nancy Roosens A1 - E. Barbau-Piednoir A1 - A. Lievens A1 - G.E. Mbongolo-Mbella A1 - D. Van Geel A1 - Els Vandermassen A1 - Amaya Leunda A1 - Sylvia Broeders A1 - Myriam Sneyers A1 - Marc Van den Bulcke KW - a KW - detection KW - Development KW - food KW - GMO KW - GMO detection KW - Molecular KW - molecular detection KW - ON KW - technology PB - WIV-ISP; PBB CY - Brussels, Belgium SN - D/2010/2505/52 U1 - 38867 ER - TY - Generic T1 - NRL-GMO: GMODetec research project (2007-2010)408 Y1 - 2010 A1 - E. Barbau-Piednoir A1 - A. Lievens A1 - Amaya Leunda A1 - Nancy Roosens A1 - Marc Van den Bulcke A1 - Myriam Sneyers A1 - Frédéric Debode A1 - Jansens,E. A1 - Berben,G. A1 - Ruttink,T. A1 - Isabel Taverniers A1 - M. De Loose KW - abstract KW - GMO KW - Project KW - Research AB - No abstract JF - LabInfo VL - 5 CP - December 2010 U1 - 38491 ER - TY - RPRT T1 - Rapport annuel du GMOLAB concernant les dossiers de validation pour l'identification des organismes génétiquement modifiés - 01/09/09 au 31/08/10 (2009-2010) Y1 - 2010 A1 - G.E. Mbongolo-Mbella A1 - Sylvia Broeders A1 - Els Vandermassen A1 - D. Van Geel A1 - Nancy Roosens A1 - E. Barbau-Piednoir A1 - Myriam Sneyers A1 - Sciacqua,M. A1 - Anne-Marie Vanherle KW - annual report KW - de KW - GMO detection KW - GMOlab KW - LE KW - qPCR KW - rapport KW - rapport annuel KW - TaqMan(r) KW - VALIDATION PB - WIV-ISP; PBB CY - Brussels, Belgium SN - D/2010/2505/51 U1 - 38813 ER - TY - RPRT T1 - Rapport annuel du GMOLAB concernant les dossiers de validation pour la quantification des organismes génétiquement modifiés - 01/09/09 au 31/08/10 (2009-2010) Y1 - 2010 A1 - G.E. Mbongolo-Mbella A1 - Sylvia Broeders A1 - Els Vandermassen A1 - D. Van Geel A1 - Nancy Roosens A1 - E. Barbau-Piednoir A1 - Myriam Sneyers A1 - Sciacqua,M. A1 - Anne-Marie Vanherle KW - de KW - GMO quantification KW - GMOlab KW - LE KW - qPCR KW - Quantification KW - rapport KW - rapport annuel KW - VALIDATION PB - WIV-ISP CY - Brussels, Belgium SN - D/2010/2505/50 U1 - 410 ER - TY - JOUR T1 - SYBR®Green qPCR screening methods for the presence of "35S promoter" and "NOS terminator" elements in food and feed products400 JF - Eur.Food.Res.Technol. Y1 - 2010 A1 - E. Barbau-Piednoir A1 - A. Lievens A1 - G.E. Mbongolo-Mbella A1 - Nancy Roosens A1 - Myriam Sneyers A1 - Amaya Leunda A1 - Marc Van den Bulcke KW - 35S promotor KW - a KW - at KW - Belgium KW - biosafety KW - Biotechnology KW - Brussels KW - crops KW - Efficiency KW - feed KW - food KW - food and feed analysis KW - genetically KW - Genetically modified KW - Genetically modified crop KW - genetically modified crops KW - GMO detection KW - health KW - Institute KW - LEVEL KW - levels KW - low level KW - M KW - method KW - methods KW - n KW - NOS terminator KW - PCR KW - Plasmids KW - PRODUCTS KW - public KW - public health KW - Public-health KW - qPCR KW - recombinant KW - SBB KW - SCREENING KW - Screening method KW - Short KW - specific KW - SYBR(r)Green KW - Target KW - Targets KW - Temperature KW - VIRUS AB - The Cauliflower Mosaic Virus "35S promotor" (p35S) and the Agrobacterium "Nopaline Synthase" terminator (tNOS) are the most represented generic recombinant elements in commercial genetically modified crops to date. A set of four new SYBR VL - 230 SN - 14382377 CP - 3 U1 - 38490 M3 - 10.1007/s00217-009-1170-5 ER - TY - JOUR T1 - A theoretical introduction to "combinatory SYBRGreen qPCR screening", a matrix-based approach for the detection of materials derived from genetically modified plants. JF - Anal Bioanal Chem Y1 - 2010 A1 - Marc Van den Bulcke A1 - Lievens, Antoon A1 - Barbau-Piednoir, Elodie A1 - Guillaume Mbongolo Mbella A1 - Nancy Roosens A1 - Myriam Sneyers A1 - Amaya Leunda KW - Algorithms KW - Animal Feed KW - Fluorescent Dyes KW - Food Analysis KW - Food, Genetically Modified KW - Models, Theoretical KW - Organic Chemicals KW - Plants, Genetically Modified KW - polymerase chain reaction AB -

The detection of genetically modified (GM) materials in food and feed products is a complex multi-step analytical process invoking screening, identification, and often quantification of the genetically modified organisms (GMO) present in a sample. "Combinatory qPCR SYBRGreen screening" (CoSYPS) is a matrix-based approach for determining the presence of GM plant materials in products. The CoSYPS decision-support system (DSS) interprets the analytical results of SYBRGREEN qPCR analysis based on four values: the C(t)- and T(m) values and the LOD and LOQ for each method. A theoretical explanation of the different concepts applied in CoSYPS analysis is given (GMO Universe, "Prime number tracing", matrix/combinatory approach) and documented using the RoundUp Ready soy GTS40-3-2 as an example. By applying a limited set of SYBRGREEN qPCR methods and through application of a newly developed "prime number"-based algorithm, the nature of subsets of corresponding GMO in a sample can be determined. Together, these analyses provide guidance for semi-quantitative estimation of GMO presence in a food and feed product.

VL - 396 CP - 6 U1 - http://www.ncbi.nlm.nih.gov/pubmed/19960341?dopt=Abstract M3 - 10.1007/s00216-009-3286-7 ER - TY - Generic T1 - Co-Extra: Europees project over co-existentie en traceerbaarheid van GGO en niet-GGO productieketens Y1 - 2009 A1 - Didier Breyer A1 - Nancy Roosens A1 - Berben,G. A1 - Isabel Taverniers A1 - Marc Van den Bulcke A1 - Myriam Sneyers KW - Co-Extra KW - coexistence KW - de KW - GMO KW - IS KW - production KW - SBB KW - SIGMEA AB - Co-Extra is een Europees project dat in april 2005 van start ging en in september 2009 wordt beëindigd. Er werkenmeer dan 200 wetenschappers aan mee die verbonden zijn aan 51 multidisciplinaire onderzoeksteams en eenaantal privé-bedrijven uit 18 landen (Europa, Brazilië, Argentinië en Rusland). Het project beschikt over een totaalbudget van 22 miljoen euro, waarvan 13 miljoen wordt betaald door de Europese Commissie via een fi nancieringsregelingvoor het 6de Europese kaderprogramma voor onderzoek.De belangrijkste doelstelling van Co-Extra bestaat erin de tools te verstrekken die nodig zijn voor de implementatievan co-existentie en traceerbaarheid met als doel de co-existentie van productieketens met GGO-producten,met gangbare producten of met producten uit de biologische landbouw te garanderen. Dit geïntegreerdeproject vormt een aanvulling op twee andere Europese projecten : SIGMEA dat vooral betrekking heeft op deco-existentie op het niveau van de landbouwproductie en Transcontainer dat zich toespitst op "biocontainment". JF - LabInfo VL - 3 CP - November 2009 U1 - 2806 ER - TY - Generic T1 - Co-Extra: Projet européen sur la coexistence et la traçabilité des filières OGM et non-OGM Y1 - 2009 A1 - Didier Breyer A1 - Nancy Roosens A1 - Berben,G. A1 - Isabel Taverniers A1 - Marc Van den Bulcke A1 - Myriam Sneyers KW - Co-Extra KW - coexistence KW - de KW - Europe KW - GMO KW - LE KW - OGM KW - production KW - programme KW - SBB KW - SIGMEA AB - Co-Extra est un projet européen qui a débuté en avril 2005 et fi nira en septembre 2009. Il regroupe plus de 200scientifi ques appartenant à 51 équipes de recherche multidisciplinaires ainsi que plusieurs compagnies privées,provenant de 18 pays (Europe, Brésil, Argentine et Russie). Le projet dispose d'un budget total de 22 millionsd'euros dont 13 millions à charge de la Commission européenne via un fi nancement lié au 6ème programme-cadrede recherche communautaire.L'objectif principal de Co-Extra est de fournir les outils nécessaires à l'implémentation de la coexistence et de latraçabilité en vue d'assurer la coexistence des fi lières utilisant des produits OGM, conventionnels ou dérivés del'agriculture biologique. Ce projet intégré complète deux autres programmes européens: SIGMEA qui porte principalementsur la coexistence au niveau de la production agricole et Transcontainer focalisé sur les méthodes debioconfi nement. JF - LabInfo VL - 3 CP - Novembre 2009 U1 - 2804 ER - TY - Generic T1 - European project Co-Extra: GM and non-GM supply chains: their co-existence and traceability Y1 - 2009 A1 - Didier Breyer A1 - Nancy Roosens A1 - Berben,G. A1 - Isabel Taverniers A1 - Marc Van den Bulcke A1 - Myriam Sneyers KW - abstract KW - co-existence KW - Co-Extra KW - coexistence KW - European KW - GM KW - GMO KW - Project KW - traceability AB -

No abstract

JF - LabInfo PB - xx CY - Brussels VL - 3 U1 - 363 ER - TY - Generic T1 - General presentation about the NRL-GMO activities since 01-07-08 Y1 - 2009 A1 - Nancy Roosens KW - Activity KW - Communication KW - general KW - GMO KW - Group KW - PRESENTATION JF - Communication group of the NRL-GMO PB - NA CY - NA CP - Communication group of the NRL-GM U1 - 378 U2 - 23/06/2009 ER - TY - Generic T1 - The use of q-PCR for GMO detection in an NRL laboratory Y1 - 2009 A1 - Nancy Roosens KW - an KW - detection KW - GMO KW - GMO detection KW - Laboratories KW - qPCR KW - use KW - Workshop JF - Workshop of the NRL-GMO PB - NA CY - NA CP - =Co-extra project committee U1 - 371 U2 - 24/11/2009 ER - TY - Generic T1 - Combinatory SYBR®Green Real-Time PCR screening as a risk management tool for low level presence of materials derived from Genetically Modified Plants384 Y1 - 2008 A1 - E. Barbau-Piednoir A1 - Els Vandermassen A1 - D. Van Geel A1 - Marc Van den Bulcke A1 - Nancy Roosens ED - Co-extra project committee KW - a KW - AS KW - Co-Extra KW - combinatory KW - conference KW - genetically KW - Genetically modified KW - genetically modified plant KW - Genetically modified plants KW - LEVEL KW - low level KW - management KW - PCR KW - plant KW - Plants KW - real time PCR KW - Real-time PCR KW - risk KW - risk management KW - SCREENING JF - Final CO-EXTRA conference CP - CO-EXTRA project comittee U1 - 384 U2 - 2008 ER - TY - Generic T1 - Effect of different storage conditions on PCR amplificability of genomic DNA extracted from pellets containing maize MON 810 maize Y1 - 2008 A1 - Piednoir,E. A1 - Els Vandermassen A1 - D. Van Geel A1 - Marc Van den Bulcke A1 - Nancy Roosens ED - Co-extra project committee KW - Co-Extra KW - conditions KW - conference KW - Dna KW - effect KW - genomic KW - maize KW - ON KW - PCR KW - STORAGE JF - Final CO-EXTRA conference CP - CO-EXTRA project comittee U1 - 391 U2 - 2008 ER - TY - JOUR T1 - Pilot market surveillance of GMM contaminations in alpha-amylase food enzyme products: a detection strategy strengthened by a newly developed qPCR method targeting a GM Bacillus licheniformis producing alpha-amylase Y1 - 0 A1 - Marie-Alice Fraiture A1 - Andrea Gobbo A1 - Chloé Guillitte A1 - U Marchesi A1 - Daniela Verginelli A1 - Joke De Greve A1 - Jolien D'aes A1 - Kevin Vanneste A1 - N. Papazova A1 - Nancy Roosens ER - TY - JOUR T1 - Whole genome sequencing en voedselveiligheid in België Y1 - 0 A1 - K. Feys A1 - N Botteldoorn A1 - V. Cantaert A1 - L. De Zutter A1 - V. Delcenserie A1 - A.G. Ameryckx A1 - J. Mahillon A1 - Marcella Mori A1 - B. Pochet A1 - Nancy Roosens A1 - Koenraad Van Hoorde A1 - Bavo Verhaegen A1 - L. Herman ER -