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Article ; Online: Comparison of 6 DNA extraction methods for isolation of high yield of high molecular weight DNA suitable for shotgun metagenomics Nanopore sequencing to detect bacteria.

Gand, Mathieu / Bloemen, Bram / Vanneste, Kevin / Roosens, Nancy H C / De Keersmaecker, Sigrid C J

2023 Volume 24, Issue 1, Page(s) 438

Abstract: 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 ...

Abstract	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.
MeSH term(s)	Metagenomics/methods ; Nanopore Sequencing ; Nanopores ; Molecular Weight ; High-Throughput Nucleotide Sequencing/methods ; DNA ; Sequence Analysis, DNA/methods ; Bacteria/genetics
Chemical Substances	DNA (9007-49-2)
Language	English
Publishing date	2023-08-04
Publishing country	England
Document type	Journal Article
ZDB-ID	2041499-7
ISSN	1471-2164 ; 1471-2164
ISSN (online)	1471-2164
ISSN	1471-2164
DOI	10.1186/s12864-023-09537-5
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Development of a portable on-site applicable metagenomic data generation workflow for enhanced pathogen and antimicrobial resistance surveillance.

Bloemen, Bram / Gand, Mathieu / Vanneste, Kevin / Marchal, Kathleen / Roosens, Nancy H C / De Keersmaecker, Sigrid C J

Scientific reports

2023 Volume 13, Issue 1, Page(s) 19656

Abstract: Rapid, accurate and comprehensive diagnostics are essential for outbreak prevention and pathogen surveillance. Real-time, on-site metagenomics on miniaturized devices, such as Oxford Nanopore Technologies MinION sequencing, could provide a promising ... ...

Abstract	Rapid, accurate and comprehensive diagnostics are essential for outbreak prevention and pathogen surveillance. Real-time, on-site metagenomics on miniaturized devices, such as Oxford Nanopore Technologies MinION sequencing, could provide a promising approach. However, current sample preparation protocols often require substantial equipment and dedicated laboratories, limiting their use. In this study, we developed a rapid on-site applicable DNA extraction and library preparation approach for nanopore sequencing, using portable devices. The optimized method consists of a portable mechanical lysis approach followed by magnetic bead-based DNA purification and automated sequencing library preparation, and resulted in a throughput comparable to a current optimal, laboratory-based protocol using enzymatic digestion to lyse cells. By using spike-in reference communities, we compared the on-site method with other workflows, and demonstrated reliable taxonomic profiling, despite method-specific biases. We also demonstrated the added value of long-read sequencing by recovering reads containing full-length antimicrobial resistance genes, and attributing them to a host species based on the additional genomic information they contain. Our method may provide a rapid, widely-applicable approach for microbial detection and surveillance in a variety of on-site settings.
MeSH term(s)	Anti-Bacterial Agents ; Workflow ; Drug Resistance, Bacterial/genetics ; Metagenome ; Metagenomics/methods ; High-Throughput Nucleotide Sequencing/methods ; DNA ; Sequence Analysis, DNA/methods ; Nanopores
Chemical Substances	Anti-Bacterial Agents ; DNA (9007-49-2)
Language	English
Publishing date	2023-11-11
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-023-46771-z
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Article ; Online: Development of a portable on-site applicable metagenomic data generation workflow for enhanced pathogen and antimicrobial resistance surveillance

Bram Bloemen / Mathieu Gand / Kevin Vanneste / Kathleen Marchal / Nancy H. C. Roosens / Sigrid C. J. De Keersmaecker

Scientific Reports, Vol 13, Iss 1, Pp 1-

2023 Volume 14

Abstract: Abstract Rapid, accurate and comprehensive diagnostics are essential for outbreak prevention and pathogen surveillance. Real-time, on-site metagenomics on miniaturized devices, such as Oxford Nanopore Technologies MinION sequencing, could provide a ... ...

Abstract	Abstract Rapid, accurate and comprehensive diagnostics are essential for outbreak prevention and pathogen surveillance. Real-time, on-site metagenomics on miniaturized devices, such as Oxford Nanopore Technologies MinION sequencing, could provide a promising approach. However, current sample preparation protocols often require substantial equipment and dedicated laboratories, limiting their use. In this study, we developed a rapid on-site applicable DNA extraction and library preparation approach for nanopore sequencing, using portable devices. The optimized method consists of a portable mechanical lysis approach followed by magnetic bead-based DNA purification and automated sequencing library preparation, and resulted in a throughput comparable to a current optimal, laboratory-based protocol using enzymatic digestion to lyse cells. By using spike-in reference communities, we compared the on-site method with other workflows, and demonstrated reliable taxonomic profiling, despite method-specific biases. We also demonstrated the added value of long-read sequencing by recovering reads containing full-length antimicrobial resistance genes, and attributing them to a host species based on the additional genomic information they contain. Our method may provide a rapid, widely-applicable approach for microbial detection and surveillance in a variety of on-site settings.
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2023-11-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Towards facilitated interpretation of shotgun metagenomics long-read sequencing data analyzed with KMA for the detection of bacterial pathogens and their antimicrobial resistance genes.

Gand, Mathieu / Navickaite, Indre / Bartsch, Lee-Julia / Grützke, Josephine / Overballe-Petersen, Søren / Rasmussen, Astrid / Otani, Saria / Michelacci, Valeria / Matamoros, Bosco Rodríguez / González-Zorn, Bruno / Brouwer, Michael S M / Di Marcantonio, Lisa / Bloemen, Bram / Vanneste, Kevin / Roosens, Nancy H C J / AbuOun, Manal / De Keersmaecker, Sigrid C J

Frontiers in microbiology

2024 Volume 15, Page(s) 1336532

Abstract: Metagenomic sequencing is a promising method that has the potential to revolutionize the world of pathogen detection and antimicrobial resistance (AMR) surveillance in food-producing environments. However, the analysis of the huge amount of data obtained ...

Abstract	Metagenomic sequencing is a promising method that has the potential to revolutionize the world of pathogen detection and antimicrobial resistance (AMR) surveillance in food-producing environments. However, the analysis of the huge amount of data obtained requires performant bioinformatics tools and databases, with intuitive and straightforward interpretation. In this study, based on long-read metagenomics data of chicken fecal samples with a spike-in mock community, we proposed confidence levels for taxonomic identification and AMR gene detection, with interpretation guidelines, to help with the analysis of the output data generated by KMA, a popular
Language	English
Publishing date	2024-04-04
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2587354-4
ISSN	1664-302X
ISSN	1664-302X
DOI	10.3389/fmicb.2024.1336532
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Article ; Online: Deepening of In Silico Evaluation of SARS-CoV-2 Detection RT-qPCR Assays in the Context of New Variants.

Gand, Mathieu / Vanneste, Kevin / Thomas, Isabelle / Van Gucht, Steven / Capron, Arnaud / Herman, Philippe / Roosens, Nancy H C / De Keersmaecker, Sigrid C J

Genes

2021 Volume 12, Issue 4

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	COVID-19/virology ; COVID-19 Nucleic Acid Testing/methods ; Computer Simulation ; Genome, Viral ; Humans ; Mutation ; RNA Probes ; Real-Time Polymerase Chain Reaction/methods ; Reverse Transcriptase Polymerase Chain Reaction/methods ; SARS-CoV-2/genetics
Chemical Substances	RNA Probes
Language	English
Publishing date	2021-04-13
Publishing country	Switzerland
Document type	Evaluation Study ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2527218-4
ISSN	2073-4425 ; 2073-4425
ISSN (online)	2073-4425
ISSN	2073-4425
DOI	10.3390/genes12040565
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Metagenomics to Detect and Characterize Viruses in Food Samples at Genome Level? Lessons Learnt from a Norovirus Study.

Buytaers, Florence E / Verhaegen, Bavo / Gand, Mathieu / D'aes, Jolien / Vanneste, Kevin / Roosens, Nancy H C / Marchal, Kathleen / Denayer, Sarah / De Keersmaecker, Sigrid C J

Foods (Basel, Switzerland)

2022 Volume 11, Issue 21

Abstract: In this proof-of-concept study on food contaminated with norovirus, we investigated the feasibility of metagenomics as a new method to obtain the whole genome sequence of the virus and perform strain level characterization but also relate to human cases ... ...

Abstract	In this proof-of-concept study on food contaminated with norovirus, we investigated the feasibility of metagenomics as a new method to obtain the whole genome sequence of the virus and perform strain level characterization but also relate to human cases in order to resolve foodborne outbreaks. We tested several preparation methods to determine if a more open sequencing approach, i.e., shotgun metagenomics, or a more targeted approach, including hybrid capture, was the most appropriate. The genetic material was sequenced using Oxford Nanopore technologies with or without adaptive sampling, and the data were analyzed with an in-house bioinformatics workflow. We showed that a viral genome sequence could be obtained for phylogenetic analysis with shotgun metagenomics if the contamination load was sufficiently high or after hybrid capture for lower contamination. Relatedness to human cases goes well beyond the results obtained with the current qPCR methods. This workflow was also tested on a publicly available dataset of food spiked with norovirus and hepatitis A virus. This allowed us to prove that we could detect even fewer genome copies and two viruses present in a sample using shotgun metagenomics. We share the lessons learnt on the satisfactory and unsatisfactory results in an attempt to advance the field.
Language	English
Publishing date	2022-10-25
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2704223-6
ISSN	2304-8158
ISSN	2304-8158
DOI	10.3390/foods11213348
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Article: Deepening of In Silico Evaluation of SARS-CoV-2 Detection RT-qPCR Assays in the Context of New Variants

Gand, Mathieu / Vanneste, Kevin / Thomas, Isabelle / Van Gucht, Steven / Capron, Arnaud / Herman, Philippe / Roosens, Nancy H. C / De Keersmaecker, Sigrid C. J

Genes. 2021 Apr. 13, v. 12, no. 4

2021

Abstract	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.
Keywords	COVID-19 infection ; RNA ; Severe acute respiratory syndrome coronavirus 2 ; computer simulation ; evolution ; genes ; oligonucleotides ; pandemic ; viruses ; China
Language	English
Dates of publication	2021-0413
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
Note	NAL-AP-2-clean
ZDB-ID	2527218-4
ISSN	2073-4425
ISSN	2073-4425
DOI	10.3390/genes12040565
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: A genoserotyping system for a fast and objective identification of Salmonella serotypes commonly isolated from poultry and pork food sectors in Belgium.

Gand, Mathieu / Mattheus, Wesley / Roosens, Nancy / Dierick, Katelijne / Marchal, Kathleen / Bertrand, Sophie / De Keersmaecker, Sigrid C J

Food microbiology

2020 Volume 91, Page(s) 103534

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Animals ; Belgium ; Decision Support Techniques ; Food Microbiology/methods ; Multiplex Polymerase Chain Reaction ; Pork Meat/microbiology ; Poultry/microbiology ; Reproducibility of Results ; Salmonella/classification ; Salmonella/genetics ; Salmonella/isolation & purification ; Serogroup ; Swine ; Time Factors
Language	English
Publishing date	2020-04-27
Publishing country	England
Document type	Journal Article
ZDB-ID	50892-5
ISSN	1095-9998 ; 0740-0020
ISSN (online)	1095-9998
ISSN	0740-0020
DOI	10.1016/j.fm.2020.103534
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Article ; Online: Use of Whole Genome Sequencing Data for a First in Silico Specificity Evaluation of the RT-qPCR Assays Used for SARS-CoV-2 Detection.

Gand, Mathieu / Vanneste, Kevin / Thomas, Isabelle / Van Gucht, Steven / Capron, Arnaud / Herman, Philippe / Roosens, Nancy H C / De Keersmaecker, Sigrid C J

International journal of molecular sciences

2020 Volume 21, Issue 15

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Betacoronavirus/genetics ; Betacoronavirus/isolation & purification ; COVID-19 ; Coronavirus Infections/diagnosis ; Coronavirus Infections/virology ; Databases, Genetic ; Genome, Viral ; Humans ; Open Reading Frames/genetics ; Pandemics ; Pneumonia, Viral/diagnosis ; Pneumonia, Viral/virology ; Polymorphism, Single Nucleotide ; RNA, Viral/analysis ; RNA, Viral/metabolism ; RNA-Dependent RNA Polymerase/genetics ; Real-Time Polymerase Chain Reaction/methods ; SARS-CoV-2 ; Sensitivity and Specificity ; Whole Genome Sequencing
Chemical Substances	RNA, Viral ; RNA-Dependent RNA Polymerase (EC 2.7.7.48)
Keywords	covid19
Language	English
Publishing date	2020-08-04
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms21155585
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Article ; Online: Assessment of the Feasibility of a Future Integrated Larger-Scale Epidemiological Study to Evaluate Health Risks of Air Pollution Episodes in Children.

Nauwelaerts, Sarah J D / De Cremer, Koen / Bustos Sierra, Natalia / Gand, Mathieu / Van Geel, Dirk / Delvoye, Maud / Vandermassen, Els / Vercauteren, Jordy / Stroobants, Christophe / Bernard, Alfred / Saenen, Nelly D / Nawrot, Tim S / Roosens, Nancy H C / De Keersmaecker, Sigrid C J

International journal of environmental research and public health

2022 Volume 19, Issue 14

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Air Pollutants/analysis ; Air Pollution/adverse effects ; Air Pollution/analysis ; Biomarkers/analysis ; Child ; Environmental Exposure/analysis ; Epidemiologic Studies ; Feasibility Studies ; Humans ; Particulate Matter/analysis
Chemical Substances	Air Pollutants ; Biomarkers ; Particulate Matter
Language	English
Publishing date	2022-07-12
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2175195-X
ISSN	1660-4601 ; 1661-7827
ISSN (online)	1660-4601
ISSN	1661-7827
DOI	10.3390/ijerph19148531
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