Publications by authors named "Paul Savelkoul"

179 Publications

External quality assessment of SARS-CoV-2-sequencing: An ESGMD-SSM pilot trial across 15 European laboratories.

J Clin Microbiol 2021 Nov 10:JCM0169821. Epub 2021 Nov 10.

Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, Netherlands.

Objective: This first pilot on external quality assessment (EQA) of SARS-CoV-2 whole genome sequencing, initiated by the ESCMID Study Group for Genomic and Molecular Diagnostics (ESGMD) and Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing.

Methods: Ten samples with varying viral loads were sent out to 15 clinical laboratories who had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centres were compared on were identification of 1) SNPs and indels, 2) Pango lineages, and 3) clusters between samples.

Results: The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to varying depth (up to 100-fold difference across centres). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignment. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data.

Conclusions: The pilot EQA was an overall success. It was able to show the high quality of participating labs and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.
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http://dx.doi.org/10.1128/JCM.01698-21DOI Listing
November 2021

Metagenomic Profiling of Fecal-Derived Bacterial Membrane Vesicles in Crohn's Disease Patients.

Cells 2021 10 19;10(10). Epub 2021 Oct 19.

Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center+, 6229 ER Maastricht, The Netherlands.

Background: In the past, many studies suggested a crucial role for dysbiosis of the gut microbiota in the etiology of Crohn's disease (CD). However, despite being important players in host-bacteria interaction, the role of bacterial membrane vesicles (MV) has been largely overlooked in the pathogenesis of CD. In this study, we addressed the composition of the bacterial and MV composition in fecal samples of CD patients and compared this to the composition in healthy individuals.

Methods: Fecal samples from six healthy subjects (HC) in addition to twelve CD patients (six active, six remission) were analyzed in this study. Fecal bacterial membrane vesicles (fMVs) were isolated by a combination of ultrafiltration and size exclusion chromatography. DNA was obtained from the fMV fraction, the pellet of dissolved feces as bacterial DNA (bDNA), or directly from feces as fecal DNA (fDNA). The fMVs were characterized by nanoparticle tracking analysis and cryo-electron microscopy. Amplicon sequencing of 16s rRNA V4 hypervariable gene regions was conducted to assess microbial composition of all fractions.

Results: Beta-diversity analysis showed that the microbial community structure of the fMVs was significantly different from the microbial profiles of the fDNA and bDNA. However, no differences were observed in microbial composition between fDNA and bDNA. The microbial richness of fMVs was significantly decreased in CD patients compared to HC, and even lower in active patients. Profiling of fDNA and bDNA demonstrated that was the most dominant phylum in these fractions, while in fMVs was dominant. In fMV, several families and genera belonging to and were significantly altered in CD patients when compared to HC.

Conclusion: The microbial alterations of MVs in CD patients particularly in and suggest a possible role of MVs in host-microbe symbiosis and induction or progression of inflammation in CD pathogenesis. Yet, the exact role for these fMV in the pathogenesis of the disease needs to be elucidated in future studies.
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http://dx.doi.org/10.3390/cells10102795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8535131PMC
October 2021

In-Depth Investigation of Conjunctival Swabs and Tear Fluid of Symptomatic COVID-19 Patients, an Observational Cohort Study.

Transl Vis Sci Technol 2021 10;10(12):32

University Eye Clinic Maastricht, Maastricht University Medical Center, Maastricht, the Netherlands.

Purpose: The putative presence of SARS-CoV-2 in ocular specimen puts healthcare workers at risk. We thoroughly examined conjunctival swabs and tear fluid in a large cohort of COVID-19 patients.

Methods: A total of 243 symptomatic laboratory-confirmed COVID-19 patients were included in this observational multicenter study. Conjunctival swabs were analyzed by reverse transcription polymerase chain reaction for detection of SARS-CoV-2 RNA. Next-generation sequencing and phylogenetic analysis were performed to identify viral strains and to determine tissue tropism. Schirmer tear samples from 43 hospitalized COVID-19 patients and 25 healthy controls were analyzed by multiplex cytokine immunoassays.

Results: Viral SARS-CoV-2 RNA was detected in conjunctival swabs from 17 (7.0%) of 243 COVID-19 patients. Conjunctival samples were positive for viral SARS-CoV-2 RNA as long as 12 days after disease onset. Cycle threshold (Ct) values for conjunctival swabs (mean 34.5 ± 5.1) were significantly higher than nasopharyngeal swabs (mean 16.7 ± 3.6). No correlation between Ct values of conjunctival and nasopharyngeal swabs was observed. The majority of positive conjunctival samples were detected only once and primarily during the first visit. Next-generation sequencing analysis revealed that the virus strain found in the conjunctiva was most often identical to the one found in the nasopharynx. Tear cytokine levels IL-1β and IL-6 were elevated in COVID-19 patients compared to healthy controls.

Conclusions: Conjunctival samples that were positive for SARS-CoV-2 RNA contained the same viral strain as the nasopharynx.

Translational Relevance: The presence of SARS-CoV-2 viral RNA and elevated cytokines in tear fluid confirm the involvement of the ocular surface in COVID-19 disease.
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http://dx.doi.org/10.1167/tvst.10.12.32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8543390PMC
October 2021

Practical and Robust NMR-Based Metabolic Phenotyping of Gut Health in Early Life.

J Proteome Res 2021 Nov 29;20(11):5079-5087. Epub 2021 Sep 29.

Department of Medical Microbiology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University, 6229 HX Maastricht, The Netherlands.

While substantial efforts have been made to optimize and standardize fecal metabolomics for studies in adults, the development of a standard protocol to analyze infant feces is, however, still lagging behind. Here, we present the development of a hands-on and robust protocol for proton H NMR spectroscopy of infant feces. The influence of extraction solvent, dilution ratio, homogenization method, filtration, and duration of centrifugation on the biochemical composition of infant feces was carefully evaluated using visual inspection of H NMR spectra in combination with multivariate statistical modeling. The optimal metabolomics protocol was subsequently applied on feces from seven infants collected at 8 weeks, 4, and 9 months of age. Interindividual variation was exceeding the variation induced by different fecal sample preparation methods, except for filtration. We recommend extracting fecal samples using water with a dilution ratio of 1:5 feces-to-water to homogenize using bead beating and to remove particulates using centrifugation. Samples collected from infants aged 8 weeks and 4 months showed elevated concentrations of milk oligosaccharide derivatives and lactic acid, whereas short-chain fatty acids (SCFAs) and branched-chain amino acids (BCAAs) were higher in the 9 month samples. The established protocol enables hands-on and robust analyses of the infant gut metabolome. The wide-ranging application of this protocol will facilitate interlaboratory comparison of infants' metabolic profiles and finally aid in a better understanding of infant gut health.
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http://dx.doi.org/10.1021/acs.jproteome.1c00617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8576838PMC
November 2021

Diagnostic and Therapeutic Considerations Towards Dientamoeba fragilis in Children: A Survey Amongst General Practitioners and Pediatricians in the Netherlands.

J Pediatr Gastroenterol Nutr 2021 Dec;73(6):e121-e125

Department of Pediatric Gastroenterology, Emma Children's Hospital Amsterdam UMC, Amsterdam.

Abstract: This survey was undertaken to obtain insight in the attitude of Dutch physicians towards pathogenicity, diagnostic- and therapeutic approach towards Dientamoeba fragilis in children. Physicians were invited by e-mail for a questionnaire. A total of 211 of 450 physicians (46.9%) completed the questionnaire, including 67 general practitioners (GPs) and 144 pediatricians. Of all respondents, 175 of 211 (82.9%) considered D fragilis a "potential pathogen", when other causes of gastro-intestinal complaints are ruled out. Only 16 of 211 (7.6%) performed diagnostic tests regularly. Diagnostic tests were performed by 162 of 211 (77%) of respondents in children with diarrhea and abdominal pain in consideration of duration of symptoms. Fecal polymerase chain reaction (PCR) was diagnostic modality of preference. Eighty-nine of 142 (62.7%) prescribed metronidazole as antibiotic of first choice. This study shows heterogeneity in clinical practice amongst Dutch physicians regarding diagnostic- and therapeutic approach of D fragilis in children. Different attitude towards pathogenicity and inconsistent guidelines could be causative factors.
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http://dx.doi.org/10.1097/MPG.0000000000003297DOI Listing
December 2021

Centre-specific bacterial pathogen typing affects infection-control decision making.

Microb Genom 2021 08;7(8)

Department of Medical Microbiology, Care and Public Health Research Institute (CAPHRI), Maastricht University Medical Center+, Maastricht, The Netherlands.

Whole-genome sequencing is becoming the standard for bacterial outbreak surveillance and infection prevention. This is accompanied by a variety of bioinformatic tools and needs bioinformatics expertise for implementation. However, little is known about the concordance of reported outbreaks when using different bioinformatic workflows. In this multi-centre proficiency testing among 13 major Dutch healthcare-affiliated centres, bacterial whole-genome outbreak analysis was assessed. Centres who participated obtained two randomized bacterial datasets of Illumina sequences, a and a Vancomycin-resistant and were asked to apply their bioinformatic workflows. Centres reported back on antimicrobial resistance, multi-locus sequence typing (MLST), and outbreak clusters. The reported clusters were analysed using a method to compare landscapes of phylogenetic trees and calculating Kendall-Colijn distances. Furthermore, fasta files were analysed by state-of-the-art single nucleotide polymorphism (SNP) analysis to mitigate the differences introduced by each centre and determine standardized SNP cut-offs. Thirteen centres participated in this study. The reported outbreak clusters revealed discrepancies between centres, even when almost identical bioinformatic workflows were used. Due to stringent filtering, some centres failed to detect extended-spectrum beta-lactamase genes and MLST loci. Applying a standardized method to determine outbreak clusters on the reported assemblies, did not result in uniformity of outbreak-cluster composition among centres.
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http://dx.doi.org/10.1099/mgen.0.000612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8549354PMC
August 2021

Harmonization of whole-genome sequencing for outbreak surveillance of and .

Microb Genom 2021 07;7(7)

Department of Medical Microbiology, Care and Public Health Research Institute (CAPHRI), Maastricht University Medical Center+, Maastricht, The Netherlands.

Whole-genome sequencing (WGS) is becoming the de facto standard for bacterial typing and outbreak surveillance of resistant bacterial pathogens. However, interoperability for WGS of bacterial outbreaks is poorly understood. We hypothesized that harmonization of WGS for outbreak surveillance is achievable through the use of identical protocols for both data generation and data analysis. A set of 30 bacterial isolates, comprising of various species belonging to the family and genera, were selected and sequenced using the same protocol on the Illumina MiSeq platform in each individual centre. All generated sequencing data were analysed by one centre using BioNumerics (6.7.3) for (i) genotyping origin of replications and antimicrobial resistance genes, (ii) core-genome multi-locus sequence typing (cgMLST) for and and whole-genome multi-locus sequencing typing (wgMLST) for all species. Additionally, a split -mer analysis was performed to determine the number of SNPs between samples. A precision of 99.0% and an accuracy of 99.2% was achieved for genotyping. Based on cgMLST, a discrepant allele was called only in 2/27 and 3/15 comparisons between two genomes, for and respectively. Based on wgMLST, the number of discrepant alleles ranged from 0 to 7 (average 1.6). For SNPs, this ranged from 0 to 11 SNPs (average 3.4). Furthermore, we demonstrate that using different assemblers to analyse the same dataset introduces up to 150 SNPs, which surpasses most thresholds for bacterial outbreaks. This shows the importance of harmonization of data-processing surveillance of bacterial outbreaks. In summary, multi-centre WGS for bacterial surveillance is achievable, but only if protocols are harmonized.
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http://dx.doi.org/10.1099/mgen.0.000567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8477410PMC
July 2021

The SARS-CoV-2 viral load in COVID-19 patients is lower on face mask filters than on nasopharyngeal swabs.

Sci Rep 2021 06 29;11(1):13476. Epub 2021 Jun 29.

inBiome B.V., Amsterdam, The Netherlands.

Face masks and personal respirators are used to curb the transmission of SARS-CoV-2 in respiratory droplets; filters embedded in some personal protective equipment could be used as a non-invasive sample source for applications, including at-home testing, but information is needed about whether filters are suited to capture viral particles for SARS-CoV-2 detection. In this study, we generated inactivated virus-laden aerosols of 0.3-2 microns in diameter (0.9 µm mean diameter by mass) and dispersed the aerosolized viral particles onto electrostatic face mask filters. The limit of detection for inactivated coronaviruses SARS-CoV-2 and HCoV-NL63 extracted from filters was between 10 to 100 copies/filter for both viruses. Testing for SARS-CoV-2, using face mask filters and nasopharyngeal swabs collected from hospitalized COVID-19-patients, showed that filter samples offered reduced sensitivity (8.5% compared to nasopharyngeal swabs). The low concordance of SARS-CoV-2 detection between filters and nasopharyngeal swabs indicated that number of viral particles collected on the face mask filter was below the limit of detection for all patients but those with the highest viral loads. This indicated face masks are unsuitable to replace diagnostic nasopharyngeal swabs in COVID-19 diagnosis. The ability to detect nucleic acids on face mask filters may, however, find other uses worth future investigation.
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http://dx.doi.org/10.1038/s41598-021-92665-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8242000PMC
June 2021

Higher Prevalence of in Crohn's Disease Exacerbations and Strain-Dependent Increase of Epithelial Resistance.

Front Microbiol 2021 8;12:598232. Epub 2021 Jun 8.

Division of Gastroenterology/Hepatology, Department of Internal Medicine, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre+, Maastricht, Netherlands.

has previously been linked to Crohn's disease (CD) exacerbations, but results are inconsistent and underlying mechanisms unknown. This study investigates the epidemiology of and its virulence factors (enterotoxin) and among 181 CD patients and the impact on the intestinal epithelial barrier . The prevalence of was significantly higher in active ( = 69/88, 78.4%) as compared to remissive ( = 58/93, 62.4%, = 0.018) CD patients. Moreover, . was associated with intestinal strictures. Interestingly, the intestinal barrier function, as examined by transepithelial electrical resistance (TEER) measurements of Caco-2 monolayers, increased when exposed to secretomes of -positive ( and isotype; increased TEER ∼160%, < 0.001) but not when exposed to negative strains. Whole metagenome sequencing and metabolomics, respectively, identified nine coding sequences and two metabolites that discriminated TEER-increasing from non-TEER-increasing strains. This study revealed a higher prevalence during exacerbation. Surprisingly, positive secretomes increased epithelial resistance, but we excluded Bft as the likely causative factor.
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http://dx.doi.org/10.3389/fmicb.2021.598232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8219053PMC
June 2021

Plant-Derived Extracellular Vesicles: Current Findings, Challenges, and Future Applications.

Membranes (Basel) 2021 May 29;11(6). Epub 2021 May 29.

Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center, 6200MD Maastricht, The Netherlands.

In recent years, plant-derived extracellular vesicles (PDEVs) have gained the interest of many experts in fields such as microbiology and immunology, and research in this field has exponentially increased. These nano-sized particles have provided researchers with a number of interesting findings, making their application in human health and disease very promising. Both in vitro and in vivo experiments have shown that PDEVs can exhibit a multitude of effects, suggesting that these vesicles may have many potential future applications, including therapeutics and nano-delivery of compounds. While the preliminary results are promising, there are still some challenges to face, such as a lack of protocol standardization, as well as knowledge gaps that need to be filled. This review aims to discuss various aspects of PDEV knowledge, including their preliminary findings, challenges, and future uses, giving insight into the complexity of conducting research in this field.
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http://dx.doi.org/10.3390/membranes11060411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8226527PMC
May 2021

Characterization of Feces-Derived Bacterial Membrane Vesicles and the Impact of Their Origin on the Inflammatory Response.

Front Cell Infect Microbiol 2021 7;11:667987. Epub 2021 May 7.

School of Nutrition and Translational Research in Metabolism (NUTRIM), Department of Medical Microbiology, Maastricht University Medical Center, Maastricht, Netherlands.

The human gastrointestinal tract harbors a diverse and complex microbiome, which interacts in a variety of ways with the host. There is compelling evidence that gut microbial dysbiosis, defined as an alteration of diversity and abundance in intestinal microbes, is an etiological factor in inflammatory bowel disease (IBD). Membrane vesicles (MVs), which are nano-sized particles released by bacteria, have been found to interact with the host and modulate the development and function of the immune system. As a result MVs have been suggested to play a critical role in both health and disease. In this study we developed a method to isolate, characterize and assess the immunoreactivity of heterogeneous populations of MVs from fecal samples (fMVs) of healthy volunteers. We successfully isolated 2*10-2*10 particles/ml from 0.5 gram of feces by using a combination of ultrafiltration and size exclusion chromatography (SEC) from 10 fecal samples. Bead-based flowcytometry in combination with tunable resistive pulse sensing (TRPS) provided a reliable method for (semi-)quantitative determination of fMVs originating from both Gram-positive and Gram-negative bacteria, while transmission electron microscopy confirmed the presence of fMVs. Real time 16s PCR on bacterial cell fractions or isolated fMVs DNA of the most common phyla () revealed differences in the relative abundance between bacteria and the fMVs. Moreover, fMVs evoke the release of TNF- by THP-1 cells in a dose-dependent matter. Also, a significant positive correlation was found between Actinobacteria/-Proteobacteria derived vesicles and the release of TNF-. It has become increasingly clear that fMVs could provide an additional layer to the definition of homeostasis or dysbiosis of the microbiota. The current study supports their potential involvement in the intestinal homeostasis or inflammatory disorders and provides putative interesting incentives for future research.
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http://dx.doi.org/10.3389/fcimb.2021.667987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8139245PMC
July 2021

Immunogenicity and safety of HBAI20 Hepatitis B vaccine in non-responders: Double-blinded, randomised, controlled phase 2 trial.

Liver Int 2021 10 24;41(10):2318-2327. Epub 2021 May 24.

Department of Medical Microbiology, School of NUTRIM, Maastricht UMC+, Maastricht, The Netherlands.

Background & Aims: Approximately 5%-10% of the general population respond inadequately to licensed recombinant hepatitis B vaccines. We assessed the immunogenicity and safety of a new HBAI20 vaccine, consisting of a new AI20 adjuvant (20-µg recombinant human IL-2 attached to 20-µg aluminium hydroxide) in combination with HBVaxPro®-10 µg.

Methods: In a double-blinded, randomised, controlled phase 2 trial, 18- to 59-year-old healthy non-responders (titre <10 mIU/ml after three or more doses of hepatitis B vaccine) were assigned (3:1 ratio) to receive either HBAI20 vaccine or HBVaxPro®-10 µg in a 0, 1 and 2-month schedule. The primary outcome was seroprotection (titre ≥ 10 mIU/ml) measured 1-3 months following the third vaccination.

Results: A total of 133 participants were randomised to receive either HBAI20 vaccine (n = 101) or HBVaxPro®-10 µg (n = 32). In the modified intention-to-treat analysis, the seroprotection rate after the third vaccination was 92.0% (80/87) in the HBAI20 group and 79.3% (23/29) in the HBVaxPro®-10-µg group, P = .068. Using a generalised linear mixed model to adjust for stratification factors, a higher odds of seroprotection with HBAI20 vaccine was shown (adjusted odds ratio = 3.48, P = .028). Frequency of mild and moderate local adverse events was greater in the HBAI20 group than in the HBVaxPro®-10 µg. Rates of severe local adverse events and systemic adverse events were low and similar in both groups.

Conclusions: In this group of hepatitis B vaccine non-responders, the HBAI20 vaccine demonstrated a higher seroprotection rate when adjusting for stratification factors and a similar safety profile compared to the licensed recombinant HBVaxPro®-10 µg.
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http://dx.doi.org/10.1111/liv.14939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8518051PMC
October 2021

Evaluation of 18 commercial serological assays for the detection of antibodies against SARS-CoV-2 in paired serum samples.

Eur J Clin Microbiol Infect Dis 2021 Aug 17;40(8):1695-1703. Epub 2021 Mar 17.

Department of Medical Microbiology, Maastricht University Medical Center Maastricht, PO 5800, 6202AZ, Maastricht, The Netherlands.

A variety of serological tests have been developed to detect the presence of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated the performance of 18 commercially available SARS-CoV-2 antibody assays. Early (6-8 days after the start of symptoms) and late sera (>14 days) from ICU patients (n=10 and n=16, respectively) and healthcare workers (n=5 and n=9, respectively) were included. Additionally, 22 sera were included to detect potential cross-reactivity. Test characteristics were determined for the 18 assays. In >14 days samples, the Vircell IgG and Wantai Ig ELISAs had superior sensitivity compared to the other ELISAs (96%). Furthermore, the Roche Ig, the Epitope Diagnostics IgM, Wantai IgM, Euroimmun IgG, and IgA all showed a specificity of 100%. The POCTs of Boson Biotech and ACRO Biotech showed the highest sensitivities: 100% and 96% (83.5-99.8), respectively. The POCT of Orient Gene Biotech, VOMED Diagnostics, and Coris-Bioconcept showed highest specificities (100%). For the IgM and IgA assays, the Euroimmun IgA test showed the highest sensitivity in early samples: 46.7% (23.5-70.9) to 53.3% (29.1-76.5). In general, all tests performed better in patients with severe symptoms (ICU patients). We conclude that the Wantai Ig and Vircell IgG ELISAs may be suitable for diagnostic purposes. The IgM/IgA tests performed poorer than their IgG/Ig counterparts but may have a role in diagnoses of SARS-CoV-2 in a population in which the background seroprevalence of IgG high, and IgM and/or IgA may distinguish between acute or past infection.
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http://dx.doi.org/10.1007/s10096-021-04220-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968571PMC
August 2021

Applying the electronic nose for pre-operative SARS-CoV-2 screening.

Surg Endosc 2021 12 2;35(12):6671-6678. Epub 2020 Dec 2.

Department of Surgery, Maastricht University Medical Center, PO Box 5800, 6202 AZ, Maastricht, The Netherlands.

Background: Infection with SARS-CoV-2 causes corona virus disease (COVID-19). The most standard diagnostic method is reverse transcription-polymerase chain reaction (RT-PCR) on a nasopharyngeal and/or an oropharyngeal swab. The high occurrence of false-negative results due to the non-presence of SARS-CoV-2 in the oropharyngeal environment renders this sampling method not ideal. Therefore, a new sampling device is desirable. This proof-of-principle study investigated the possibility to train machine-learning classifiers with an electronic nose (Aeonose) to differentiate between COVID-19-positive and negative persons based on volatile organic compounds (VOCs) analysis.

Methods: Between April and June 2020, participants were invited for breath analysis when a swab for RT-PCR was collected. If the RT-PCR resulted negative, the presence of SARS-CoV-2-specific antibodies was checked to confirm the negative result. All participants breathed through the Aeonose for five minutes. This device contains metal-oxide sensors that change in conductivity upon reaction with VOCs in exhaled breath. These conductivity changes are input data for machine learning and used for pattern recognition. The result is a value between - 1 and + 1, indicating the infection probability.

Results: 219 participants were included, 57 of which COVID-19 positive. A sensitivity of 0.86 and a negative predictive value (NPV) of 0.92 were found. Adding clinical variables to machine-learning classifier via multivariate logistic regression analysis, the NPV improved to 0.96.

Conclusions: The Aeonose can distinguish COVID-19 positive from negative participants based on VOC patterns in exhaled breath with a high NPV. The Aeonose might be a promising, non-invasive, and low-cost triage tool for excluding SARS-CoV-2 infection in patients elected for surgery.
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http://dx.doi.org/10.1007/s00464-020-08169-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709806PMC
December 2021

Intestinal Microbiota in Children With Symptomatic Dientamoeba fragilis Infection: A Case-control Study.

Pediatr Infect Dis J 2021 04;40(4):279-283

Department of Pediatric Gastroenterology, Emma Children's Hospital, Amsterdam UMC, Amsterdam, The Netherlands.

Background: Dientamoeba fragilis in children has been associated with gastrointestinal symptoms, like abdominal pain and diarrhea. The mechanism underlying these symptoms in children with D. fragilis remains unclear. We hypothesized that concomitant microbial alterations, which have been described in other parasitic infections, may be associated with gastrointestinal symptoms in D. fragilis.

Methods: In this case-control study performed in 2 centers, 19 children referred to a pediatrician because of gastrointestinal symptoms and with a positive fecal PCR for D. fragilis were included as cases. We included 19 healthy children as controls and matched for age and gender, selected from an existing cohort of 63 children. A PCR for D. fragilis was performed on fecal samples of the 19 controls to assess D. fragilis carriership in this asymptomatic group. Microbiota was analyzed with the IS-pro technique, and the intestinal microbiota composition and diversity were compared between the 2 groups.

Results: Microbiota of children with D. fragilis and gastrointestinal symptoms did not significantly differ in terms of composition and diversity compared with controls, both on phylum and species level. In the asymptomatic controls, a positive fecal PCR for D. fragilis was found in 16 of 19 (84.2%).

Conclusion: Intestinal microbiota does not seem to play a key role in the presence of clinical symptoms in children with D. fragilis. The pathogenicity of D. fragilis and pathophysiologic pathways underlying the development of gastrointestinal symptoms remains yet to be clarified.
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http://dx.doi.org/10.1097/INF.0000000000002975DOI Listing
April 2021

Workload, diagnostic work-up and treatment of urinary tract infections in adults during out-of-hours primary care: a retrospective cohort study.

BMC Fam Pract 2020 11 10;21(1):231. Epub 2020 Nov 10.

Department of Family Medicine, CAPHRI Care and Public Health Research Institute, Maastricht University Medical Centre, P.O. Box 616, Maastricht, MD, 6200, The Netherlands.

Background: Urinary tract infections (UTIs) are one of the most common infections in primary care. Previous research showed that GPs find it challenging to diagnose UTIs and frequently divert from guidelines leading to unwarranted antibiotic prescriptions and inefficient use of diagnostics such as urinary cultures. We hypothesise that management of UTIs during out-of-hours care may be extra challenging due to a higher workload and logistical issues regarding diagnostic work-up and obtaining results. We therefore aimed to study the workload, diagnostic work-up and treatment of UTIs during out-of-hours primary care.

Methods: We performed a retrospective observational cohort study in which we analysed a full year (2018) of electronic patient records of two large Dutch GP out-of-hours centres. All adult patients with UTI symptoms were included in this study. Descriptive statistics and multivariate regression were used to analyse diagnostics and subsequent management.

Results: A total of 5657 patients were included (78.9% female, mean age of 54 years), with an average of eight patients per day that contact a GP out-of-hours centre because of UTI symptoms. Urinary dipsticks were used in 87.5% of all patients visiting the out-of-hours centres with UTI symptoms. Strikingly, urinary cultures were only requested in 10.3% of patients in which urinary culture was indicated. Seventy-four percent of the patients received antibiotics. Seventy-nine percent of the patients with a negative nitrite test still received antibiotics. Remarkably, patients at risk of complications because of a UTI, such as men, received fewer antibiotic prescriptions.

Conclusions: In total, 74% of the patients received antibiotics. 8 out of 10 patients still received an antibiotic prescription in case of a negative nitrite test, and 9 out of 10 patients with an indication did not receive a urine culture. In conclusion, we found that correctly diagnosing UTIs and prescribing antibiotics for UTIs is a challenge that needs major improvement, especially during out-of-hours GP care.
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http://dx.doi.org/10.1186/s12875-020-01305-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653778PMC
November 2020

Influence of probiotic supplementation on the developing microbiota in human preterm neonates.

Gut Microbes 2020 11;12(1):1-16

Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University , Maastricht, The Netherlands.

Background: Oral administration of probiotic bacteria to preterm neonates has been recommended to prevent the development of necrotizing enterocolitis (NEC). The influence of probiotics on the endogenous microbiome, however, has remained incompletely understood.

Study Design & Methods: Here, we performed an observational study including 80 preterm neonates born at a gestational age <32-weeks to characterize the persistence of probiotic bacteria after no treatment or oral administration of two different probiotic formula and their influence on the microbial ecosystem during and after the intervention and their association with the development of NEC. Weekly fecal samples were profiled by 16S rRNA sequencing and monitored for the presence of the probiotic bacteria by quantitative PCR.

Results: Microbiota profiles differed significantly between the control group and both probiotic groups. Probiotic supplementation was associated with lower temporal variation as well as higher relative abundance of and combined with reduced abundance of , and . Colonization by probiotic bifidobacteria was observed in approximately 50% of infants although it remained transient in the majority of cases. A significantly reduced monthly incidence of NEC was observed in neonates supplemented with probiotics.

Conclusion: Our results demonstrate successful transient colonization by probiotic bacteria and a significant influence on the endogenous microbiota with a reduced abundance of bacterial taxa associated with the development of NEC. These results emphasize that probiotic supplementation may allow targeted manipulation of the enteric microbiota and confer a clinical benefit. (Clinical Trial Registry accession number: DRKS/GCTR 00021034).
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http://dx.doi.org/10.1080/19490976.2020.1826747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588225PMC
November 2020

Serial measurements in COVID-19-induced acute respiratory disease to unravel heterogeneity of the disease course: design of the Maastricht Intensive Care COVID cohort (MaastrICCht).

BMJ Open 2020 09 29;10(9):e040175. Epub 2020 Sep 29.

Department of Intensive Care, Maastricht University Medical Center+, Maastricht, The Netherlands.

Introduction: The course of the disease in SARS-CoV-2 infection in mechanically ventilated patients is unknown. To unravel the clinical heterogeneity of the SARS-CoV-2 infection in these patients, we designed the prospective observational Maastricht Intensive Care COVID cohort (MaastrICCht). We incorporated serial measurements that harbour aetiological, diagnostic and predictive information. The study aims to investigate the heterogeneity of the natural course of critically ill patients with a SARS-CoV-2 infection.

Methods And Analysis: Mechanically ventilated patients admitted to the intensive care with a SARS-CoV-2 infection will be included. We will collect clinical variables, vital parameters, laboratory variables, mechanical ventilator settings, chest electrical impedance tomography, ECGs, echocardiography as well as other imaging modalities to assess heterogeneity of the course of a SARS-CoV-2 infection in critically ill patients. The MaastrICCht is also designed to foster various other studies and registries and intends to create an open-source database for investigators. Therefore, a major part of the data collection is aligned with an existing national intensive care data registry and two international COVID-19 data collection initiatives. Additionally, we create a flexible design, so that additional measures can be added during the ongoing study based on new knowledge obtained from the rapidly growing body of evidence. The spread of the COVID-19 pandemic requires the swift implementation of observational research to unravel heterogeneity of the natural course of the disease of SARS-CoV-2 infection in mechanically ventilated patients. Our study design is expected to enhance aetiological, diagnostic and prognostic understanding of the disease. This paper describes the design of the MaastrICCht.

Ethics And Dissemination: Ethical approval has been obtained from the medical ethics committee (Medisch Ethische Toetsingscommissie 2020-1565/3 00 523) of the Maastricht University Medical Centre+ (Maastricht UMC+), which will be performed based on the Declaration of Helsinki. During the pandemic, the board of directors of Maastricht UMC+ adopted a policy to inform patients and ask their consent to use the collected data and to store serum samples for COVID-19 research purposes. All study documentation will be stored securely for fifteen years after recruitment of the last patient. The results will be published in peer-reviewed academic journals, with a preference for open access journals, while particularly considering deposition of the manuscripts on a preprint server early.

Trial Registration Number: The Netherlands Trial Register (NL8613).
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http://dx.doi.org/10.1136/bmjopen-2020-040175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526030PMC
September 2020

How to Count Our Microbes? The Effect of Different Quantitative Microbiome Profiling Approaches.

Front Cell Infect Microbiol 2020 7;10:403. Epub 2020 Aug 7.

Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Center+, Maastricht, Netherlands.

Next-generation sequencing (NGS) has instigated the research on the role of the microbiome in health and disease. The compositional nature of such microbiome datasets makes it however challenging to identify those microbial taxa that are truly associated with an intervention or health outcome. Quantitative microbiome profiling overcomes the compositional structure of microbiome sequencing data by integrating absolute quantification of microbial abundances into the NGS data. Both cell-based methods (e.g., flow cytometry) and molecular methods (qPCR) have been used to determine the absolute microbial abundances, but to what extent different quantification methods generate similar quantitative microbiome profiles has so far not been explored. Here we compared relative microbiome profiling (without incorporation of microbial quantification) to three variations of quantitative microbiome profiling: (1) microbial cell counting using flow cytometry (QMP), (2) counting of microbial cells using flow cytometry combined with Propidium Monoazide pre-treatment of fecal samples before metagenomics DNA isolation in order to only profile the microbial composition of intact cells (QMP-PMA), and (3) molecular based quantification of the microbial load using qPCR targeting the 16S rRNA gene. Although qPCR and flow cytometry both resulted in accurate and strongly correlated results when quantifying the bacterial abundance of a mock community of bacterial cells, the two methods resulted in highly divergent quantitative microbial profiles when analyzing the microbial composition of fecal samples from 16 healthy volunteers. These differences could not be attributed to the presence of free extracellular prokaryotic DNA in the fecal samples as sample pre-treatment with Propidium Monoazide did not improve the concordance between qPCR-based and flow cytometry-based QMP. Also lack of precision of qPCR was ruled out as a major cause of the disconcordant findings, since quantification of the fecal microbial load by the highly sensitive digital droplet PCR correlated strongly with qPCR. In conclusion, quantitative microbiome profiling is an elegant approach to bypass the compositional nature of microbiome NGS data, however it is important to realize that technical sources of variability may introduce substantial additional bias depending on the quantification method being used.
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http://dx.doi.org/10.3389/fcimb.2020.00403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426659PMC
June 2021

Re: 'The renal safety of a single dose of gentamicin in patients with sepsis in the emergency department' - Author's reply.

Clin Microbiol Infect 2021 02 19;27(2):301-302. Epub 2020 Aug 19.

Department of Internal Medicine, Division of General Medicine, Section of Acute Medicine, Maastricht University Medical Center, Maastricht, the Netherlands; CAPHRI School for Public Health and Primary Care, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, the Netherlands.

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http://dx.doi.org/10.1016/j.cmi.2020.08.016DOI Listing
February 2021

Prevalence and abundance of selected genes conferring macrolide resistance genes in COPD patients during maintenance treatment with azithromycin.

Antimicrob Resist Infect Control 2020 07 28;9(1):116. Epub 2020 Jul 28.

Laboratory for Microbiology and Infection Control, Amphia Hospital, Breda, The Netherlands.

Objectives: Maintenance treatment with macrolide antibiotics has shown to be effective in reducing exacerbations in COPD patients. A major concern with prolonged treatment with antibiotics is the development of bacterial resistance. In this study we determined the effect of azithromycin on the development and acquisition of resistance to macrolides in the nasopharyngeal flora in COPD patients.

Methods: This study was part of the COLUMBUS trial, a randomised, double-blind, placebo-controlled trial to measure the effect of maintenance treatment with azithromycin in 92 COPD patients on the exacerbation rates during a 12-month period. In order to determine resistance to macrolides, we used a targeted metagenomic approach to measure the presence and relative abundance of specific macrolide resistance genes ermB, ermF and mefA in throat samples collected at different time-points during this 12-month period.

Results: There was no increased risk for acquisition of macrolide resistance genes in the azithromycin group compared to the placebo group in COPD patients. However, loss of the macrolide resistance gene ermB was increased overtime in the placebo treated group compared to the azithromycin group (n = 5 for the placebo group versus n = 0 for the azithromycin group at 12 months; p = 0.012). The change in relative abundance of the three macrolide-resistance genes showed that all but one (ermF) increased during treatment with azithromycin.

Conclusions: The acquisition rate of macrolide resistance genes in COPD patients treated with azithromycin maintenance therapy was limited, but the relative abundance of macrolide resistance genes increased significantly over time compared to placebo. This study was part of the COLUMBUS trial ( Clinicaltrials.gov , NCT00985244 ).
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http://dx.doi.org/10.1186/s13756-020-00783-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7389634PMC
July 2020

Effects of Antibiotics on the Intestinal Microbiota of Mice.

Antibiotics (Basel) 2020 Apr 17;9(4). Epub 2020 Apr 17.

Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark.

Studies on human and mouse gastrointestinal microbiota have correlated the composition of the microbiota to a variety of diseases, as well as proved it vital to prevent colonization with resistant bacteria, a phenomenon known as colonization resistance. Antibiotics dramatically modify the gut community and there are examples of how antibiotic usage lead to colonization with resistant bacteria [e.g., dicloxacillin usage selecting for ESBL-producing carriage], as shown by Hertz et al. Here, we investigated the impact of five antibiotics [cefotaxime, cefuroxime, dicloxacillin, clindamycin, and ciprofloxacin] on the intestinal microbiota in mice. Five different antibiotics were each given to groups of five mice. The intestinal microbiotas were profiled by use of the IS-pro analysis; a 16S-23S rDNA interspace [IS]-region-based profiling method. For the mice receiving dicloxacillin and clindamycin, we observed dramatic shifts in dominating phyla from day 1 to day 5. Of note, diversity increased, but overall bacterial load decreased. For ciprofloxacin, cefotaxime, and cefuroxime there were few overall changes. We speculate that antibiotics with efficacy against the abundant anaerobes in the gut, particularly Bacteroidetes, can in fact be selected for resistant bacteria, disregarding the spectrum of activity.
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http://dx.doi.org/10.3390/antibiotics9040191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7235770PMC
April 2020

Dientamoeba fragilis in children: a systematic review on diagnostic considerations and efficacy of treatment.

Expert Rev Gastroenterol Hepatol 2020 Apr 21;14(4):231-242. Epub 2020 Mar 21.

Department of Pediatrics, Tergooi Hospital, Blaricum, The Netherlands.

: The presence of in feces is characterized by an asymptomatic carrier ship to a spectrum of gastrointestinal symptoms. However, a causal relationship remains to be elucidated. In this systematic review, we aimed to evaluate the relationship between the eradication of and symptoms to establish the strength of evidence that in symptomatic children warrants antibiotic treatment.: This systematic review covers a challenge in daily clinical practice. Is it necessary to test for in children with gastrointestinal symptoms and does a positive fecal PCR test warrant treatment?: Testing for seems justified in a selection of children with persistent unexplained chronic abdominal pain and diarrhea. Treatment of should be withhold until other causes like celiac disease have been excluded. Both microscopic and Real Time-PCR methods (or a combination of the two) can be used for diagnosis. Paromomycin or clioquinol are antibiotics of choice based on their small spectrum of activity, fewer side effects, and better eradication rates than metronidazole. Future randomized studies, with strict inclusion criteria, appropriate diagnostic testing, and doses of antibiotics based on bodyweight are warranted.
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http://dx.doi.org/10.1080/17474124.2020.1739520DOI Listing
April 2020

Neonatal microbiota development and the effect of early life antibiotics are determined by two distinct settler types.

PLoS One 2020 5;15(2):e0228133. Epub 2020 Feb 5.

Department of Pediatrics, St Antonius Hospital, Nieuwegein, The Netherlands.

The neonatal period, during which the initial gut microbiota is acquired, is a critical phase. The healthy development of the infant's microbiome can be interrupted by external perturbations, like antibiotics, which are associated with profound effects on the gut microbiome and various disorders later in life. The aim of this study was to investigate the development of intestinal microbiota and the effect of antibiotic exposure during the first three months of life in term infants. Fecal samples were collected from healthy infants and infants who received antibiotics in the first week of life at one week, one month, and three months after birth. Microbial composition was analyzed using IS-pro and compared between antibiotics-treated and untreated infants. In total, 98 infants, divided into four groups based on feeding type and delivery mode, were analyzed. At one week of age, samples clustered into two distinct groups, which were termed "settler types", based on their Bacteroidetes abundance. Caesarean-born infants belonged to the low-Bacteroidetes settler type, but vaginally-born infants were divided between the two groups. The antibiotics effect was assessed within a subgroup of 45 infants, vaginally-born and exclusively breastfed, to minimize the effect of other confounders. Antibiotics administration resulted in lower Bacteroidetes diversity and/or a delay in Bacteroidetes colonization, which persisted for three months, and in a differential development of the microbiota. Antibiotics resulted in pronounced effects on the Bacteroidetes composition and dynamics. Finally, we hypothesize that stratification of children's cohorts based on settler types may reveal group effects that might otherwise be masked.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228133PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001974PMC
April 2020

Development of the Microbiota and Associations With Birth Mode, Diet, and Atopic Disorders in a Longitudinal Analysis of Stool Samples, Collected From Infancy Through Early Childhood.

Gastroenterology 2020 05 18;158(6):1584-1596. Epub 2020 Jan 18.

School of Nutrition and Translational Research in Metabolism (NUTRIM), Department of Medical Microbiology, Maastricht University Medical Centre, Maastricht, the Netherlands; School for Public Health and Primary Care (Caphri), Department of Medical Microbiology, Maastricht University Medical Centre, Maastricht, the Netherlands; in Vivo Planetary Health: an affiliate of the World Universities Network (WUN), West New York, New Jersey. Electronic address:

Background & Aims: Establishment of the gastrointestinal microbiota during infancy affects immune system development and oral tolerance induction. Perturbations in the microbiome during this period can contribute to development of immune-mediated diseases. We monitored microbiota maturation and associations with subsequent development of allergies in infants and children.

Methods: We collected 1453 stool samples, at 5, 13, 21, and 31 weeks postpartum (infants), and once at school age (6-11 years), from 440 children (49.3% girls, 24.8% born by cesarean delivery; all children except for 6 were breastfed for varying durations; median 40 weeks; interquartile range, 30-53 weeks). Microbiota were analyzed by amplicon sequencing. Children were followed through 3 years of age for development of atopic dermatitis; data on allergic sensitization and asthma were collected when children were school age.

Results: Diversity of fecal microbiota, assessed by Shannon index, did not differ significantly among children from 5 through 13 weeks after birth, but thereafter gradually increased to 21 and 31 weeks. Most bacteria within the Bacteroidetes and Proteobacteria phyla were already present at 5 weeks after birth, whereas many bacteria of the Firmicutes phylum were acquired at later times in infancy. At school age, many new Actinobacteria, Firmicutes, and Bacteroidetes bacterial taxa emerged. The largest increase in microbial diversity occurred after 31 weeks. Vaginal, compared with cesarean delivery, was most strongly associated with an enrichment of Bacteroides species at 5 weeks through 31 weeks. From 13 weeks onward, diet became the most important determinant of microbiota composition; cessation of breastfeeding, rather than solid food introduction, was associated with changes. For example, Bifidobacteria, staphylococci, and streptococci significantly decreased on cessation of breastfeeding, whereas bacteria within the Lachnospiraceae family (Pseudobutyrivibrio, Lachnobacterium, Roseburia, and Blautia) increased. When we adjusted for confounding factors, we found fecal microbiota composition to be associated with development of atopic dermatitis, allergic sensitization, and asthma. Members of the Lachnospiraceae family, as well as the genera Faecalibacterium and Dialister, were associated with a reduced risk of atopy.

Conclusions: In a longitudinal study of fecal microbiota of children from 5 weeks through 6 to 11 years, we tracked changes in diversity and composition associated with the development of allergies and asthma.
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http://dx.doi.org/10.1053/j.gastro.2020.01.024DOI Listing
May 2020

Decreasing prevalence of contamination with extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) in retail chicken meat in the Netherlands.

PLoS One 2019 31;14(12):e0226828. Epub 2019 Dec 31.

Department of Infection Control, Amphia Hospital, Breda, the Netherlands.

Retail chicken meat is a potential source of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E). In the past decade, vast national efforts were undertaken to decrease the antibiotic use in the veterinary sector, resulting in a 58% decrease in antibiotic sales in the sector between 2009 and 2014. This decrease in antibiotic use was followed by a decrease in ESBL-E prevalence in broilers. The current study investigates the prevalence of contamination with ESBL-E in retail chicken meat purchased in the Netherlands between December 2013 and August 2015. It looks at associations between the prevalence of contamination with ESBL-E and sample characteristics such as method of farming (free-range or conventional), supermarket chain of purchase and year of purchase. In the current study, 352 chicken meat samples were investigated for the presence of ESBL-E using selective culture methods. Six samples were excluded due to missing isolates or problems obtaining a good quality sequence leaving 346 samples for further analyses. Of these 346 samples, 188 (54.3%) were positive for ESBL-E, yielding 216 ESBL-E isolates (Escherichia coli (n = 204), Klebsiella pneumoniae (n = 11) and Escherichia fergusonii (n = 1)). All ESBL-E isolates were analysed using whole-genome sequencing. The prevalence of contamination with ESBL-E in retail chicken meat decreased from 68.3% in 2014 to 44.6% in 2015, absolute risk difference 23.7% (95% confidence interval (CI): 12.6% - 34.1%). The ESBL-E prevalence was lower in free-range chicken meat (36.4%) compared with conventional chicken meat (61.5%), absolute risk difference 25.2% (95% CI: 12.9% - 36.5%). The prevalence of contamination with ESBL-E varied between supermarket chains, the highest prevalence of contamination was found in supermarket chain 4 (76.5%) and the lowest in supermarket chain 1 (37.8%). Pairwise isolate comparisons using whole-genome multilocus sequence typing (wgMLST) showed that clustering of isolates occurs more frequently within supermarket chains than between supermarket chains. In conclusion, the prevalence of contamination with ESBL-E in retail chicken in the Netherlands decreased over time; nevertheless, it remains substantial and as such a potential source for ESBL-E in humans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226828PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938319PMC
April 2020

Gut microbiota in wheezing preschool children and the association with childhood asthma.

Allergy 2020 06 29;75(6):1473-1476. Epub 2020 Jan 29.

Department of Medical Microbiology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University Medical Centre+, Maastricht, The Netherlands.

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http://dx.doi.org/10.1111/all.14156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317729PMC
June 2020

Cell Type- and Exposure-Specific Modulation of CD63/CD81-Positive and Tissue Factor-Positive Extracellular Vesicle Release in response to Respiratory Toxicants.

Oxid Med Cell Longev 2019 14;2019:5204218. Epub 2019 Aug 14.

Department of Medical Microbiology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, PO box 5800, 6202AZ Maastricht, Netherlands.

Chronic exposure to respiratory stressors increases the risk for pulmonary and cardiovascular diseases. Previously, we have shown that cigarette smoke extract (CSE) triggers the release of CD63CD81 and tissue factor (TF) procoagulant extracellular vesicles (EVs) by bronchial epithelial cells via depletion of cell surface thiols. Here, we hypothesized that this represents a universal response for different pulmonary cell types and respiratory exposures. Using bead-based flow cytometry, we found that bronchial epithelial cells and pulmonary fibroblasts, but not pulmonary microvascular endothelial cells or macrophages, release CD63CD81 and TF EVs in response to CSE. Cell surface thiols decreased in all cell types upon CSE exposure, whereas depletion of cell surface thiols using bacitracin only triggered EV release by epithelial cells and fibroblasts. The thiol-antioxidant NAC prevented the EV induction by CSE in epithelial cells and fibroblasts. Exposure of epithelial cells to occupational silica nanoparticles and particulate matter (PM) from outdoor air pollution also enhanced EV release. Cell surface thiols were mildly decreased and NAC partly prevented the EV induction for PM, but not for silica and PM. Taken together, induction of procoagulant EVs is a cell type-specific response to CSE. Moreover, induction of CD63CD81 and TF EVs in bronchial epithelial cells appears to be a universal response to various respiratory stressors. TF EVs may serve as biomarkers of exposure and/or risk in response to respiratory exposures and may help to guide preventive treatment decisions.
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http://dx.doi.org/10.1155/2019/5204218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6710792PMC
January 2020

Contact precautions in single-bed or multiple-bed rooms for patients with extended-spectrum β-lactamase-producing Enterobacteriaceae in Dutch hospitals: a cluster-randomised, crossover, non-inferiority study.

Lancet Infect Dis 2019 10 23;19(10):1069-1079. Epub 2019 Aug 23.

Department of Infection Control, Amphia Hospital, Breda, Netherlands; Microvida Laboratory for Microbiology, Amphia Hospital, Breda, Netherlands; Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, University of Utrecht, Utrecht, Netherlands.

Background: Use of single-bed rooms for control of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is under debate; the added value when applying contact precautions has not been shown. We aimed to assess whether an isolation strategy of contact precautions in a multiple-bed room was non-inferior to a strategy of contact precautions in a single-bed room for preventing transmission of ESBL-producing Enterobacteriaceae.

Methods: We did a cluster-randomised, crossover, non-inferiority study on medical and surgical wards of 16 Dutch hospitals. During two consecutive study periods, either contact precautions in a single-bed room or contact precautions in a multiple-bed room were applied as the preferred isolation strategy for patients with ESBL-producing Enterobacteriaceae cultured from a routine clinical sample (index patients). Eligible index patients were aged 18 years or older, had no strict indication for barrier precautions in a single-bed room, had a culture result reported within 7 days of culture and before discharge, and had no wardmate known to be colonised or infected with an ESBL-producing Enterobacteriaceae isolate of the same bacterial species with a similar antibiogram. Hospitals were randomly assigned in a 1:1 ratio by computer to one of two sequences of isolation strategies, stratified by university or non-university hospital. Allocation was masked for laboratory technicians who assessed the outcomes but not for patients, treating doctors, and infection-control practitioners enrolling index patients. The primary outcome was transmission of ESBL-producing Enterobacteriaceae to wardmates, which was defined as rectal carriage of an ESBL-producing Enterobacteriaceae isolate that was clonally related to the index patient's isolate in at least one wardmate. The primary analysis was done in the per-protocol population, which included patients who were adherent to the assigned room type. A 10% non-inferiority margin for the risk difference was used to assess non-inferiority. This study is registered with Nederlands Trialregister, NTR2799.

Findings: 16 hospitals were randomised, eight to each sequence of isolation strategies. All hospitals randomised to the sequence single-bed room then multiple-bed room and five of eight hospitals randomised to the sequence multiple-bed room then single-bed room completed both study periods and were analysed. From April 24, 2011, to Feb 27, 2014, 1652 index patients and 12 875 wardmates were assessed for eligibility. Of those, 693 index patients and 9527 wardmates were enrolled and 463 index patients and 7093 wardmates were included in the per-protocol population. Transmission of ESBL-producing Enterobacteriaceae to at least one wardmate was identified for 11 (4%) of 275 index patients during the single-bed room strategy period and for 14 (7%) of 188 index patients during the multiple-bed room strategy period (crude risk difference 3·4%, 90% CI -0·3 to 7·1).

Interpretation: For patients with ESBL-producing Enterobacteriaceae cultured from a routine clinical sample, an isolation strategy of contact precautions in a multiple-bed room was non-inferior to a strategy of contact precautions in a single-bed room for preventing transmission of ESBL-producing Enterobacteriaceae. Non-inferiority of the multiple-bed room strategy might change the current single-bed room preference for isolation of patients with ESBL-producing Enterobacteriaceae and, thus, broaden infection-control options for ESBL-producing Enterobacteriaceae in daily clinical practice.

Funding: Netherlands Organisation for Health Research and Development.
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http://dx.doi.org/10.1016/S1473-3099(19)30262-2DOI Listing
October 2019

Moderate positive predictive value of a multiplex real-time PCR on whole blood for pathogen detection in critically ill patients with sepsis.

Eur J Clin Microbiol Infect Dis 2019 Oct 26;38(10):1829-1836. Epub 2019 Jun 26.

Department of Intensive Care Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.

A novel multiplex real-time PCR for bloodstream infections (BSI-PCR) detects pathogens directly in blood. This study aimed at determining the positive predictive value (PPV) of BSI-PCR in critically ill patients with sepsis. We included consecutive patients with presumed sepsis upon admission to the intensive care unit (ICU). The multiplexed BSI-PCR included 17 individual PCRs for a broad panel of species- and genus-specific DNA targets. BSI-PCR results were compared with a reference diagnosis for which plausibility of infection and causative pathogen(s) had been prospectively assessed by trained observers, based on available clinical and microbiological evidence. PPV and false positive proportion (FPP) were calculated. Clinical plausibility of discordant positive results was adjudicated by an expert panel. Among 325 patients, infection likelihood was categorized as confirmed, uncertain, and ruled out in 210 (65%), 88 (27%), and 27 (8%) subjects, respectively. BSI-PCR identified one or more microorganisms in 169 (52%) patients, of whom 104 (61%) had at least one detection in accordance with the reference diagnosis. Discordant positive PCR results were observed in 95 patients, including 30 subjects categorized as having an "unknown" pathogen. Based on 5525 individual PCRs yielding 295 positive results, PPV was 167/295 (57%) and FPP was 128/5525 (2%). Expert adjudication of the 128 discordant PCR findings resulted in an adjusted PPV of 68% and FPP of 2%. BSI-PCR was all-negative in 156 patients, including 79 (51%) patients in whom infection was considered ruled out. BSI-PCR may complement conventional cultures and expedite the microbiological diagnosis of sepsis in ICU patients, but improvements in positive predictive value of the test are warranted before its implementation in clinical practice can be considered.
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http://dx.doi.org/10.1007/s10096-019-03616-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778535PMC
October 2019
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