Publications by authors named "Els Wessels"

24 Publications

  • Page 1 of 1

Direct-Acting Antiviral Treatment for Hepatitis C Genotypes Uncommon in High-Income Countries: A Dutch Nationwide Cohort Study.

Open Forum Infect Dis 2021 Feb 6;8(2):ofab006. Epub 2021 Jan 6.

Department of Medical Microbiology, Section of Clinical Virology, Amsterdam Infection & Immunity Institute, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands.

Background: The majority of hepatitis C virus (HCV) infections are found in low- and middle-income countries, which harbor many region-specific HCV subtypes. Nevertheless, direct-acting antiviral (DAA) trials have almost exclusively been conducted in high-income countries, where mainly epidemically spread HCV subtypes are present. Recently, several studies have demonstrated suboptimal DAA efficacy for certain nonepidemic subtypes, which could hamper global HCV elimination. Therefore, we aimed to evaluate DAA efficacy in patients treated for a nonepidemic HCV genotype infection in the Netherlands.

Methods: We performed a nationwide retrospective study including patients treated with interferon-free DAAs for an HCV genotype other than 1a/1b/2a/2b/3a/4a/4d. The genotype was determined by NS5B region phylogenetic analysis. The primary end point was SVR-12. If stored samples were available, NS5A and NS5B sequences were obtained for resistance-associated substitutions (RAS) evaluation.

Results: We included 160 patients, mainly infected with nonepidemic genotype 2 (41%) and 4 (31%) subtypes. Most patients were from Africa (45%) or South America (24%); 51 (32%) were cirrhotic. SVR-12 was achieved in 92% (140/152) of patients with available SVR-12 data. Only 73% (8/11) genotype 3-infected patients achieved SVR-12, the majority being genotype 3b patients with 63% (5/8) SVR. Regardless of SVR, all genotype 3b patients had 30K and 31M RAS.

Conclusions: The DAA efficacy we observed in most nonepidemic genotypes in the Netherlands seems reassuring. However, the low SVR-12 rate in subtype 3b infections is alarming, especially as it is common in several HCV-endemic countries. Alongside earlier results, our results indicate that a remaining challenge for global HCV elimination is confirming and monitoring DAA efficacy in nonepidemic genotypes.
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http://dx.doi.org/10.1093/ofid/ofab006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881754PMC
February 2021

The end of the laboratory developed test as we know it? Recommendations from a national multidisciplinary taskforce of laboratory specialists on the interpretation of the IVDR and its complications.

Clin Chem Lab Med 2020 Nov 23. Epub 2020 Nov 23.

Department of Clinical Chemistry, Zuyderland Medical Centre, Heerlen, The Netherlands.

The in vitro diagnostic medical devices regulation (IVDR) will take effect in May 2022. This regulation has a large impact on both the manufacturers of in vitro diagnostic medical devices (IVD) and clinical laboratories. For clinical laboratories, the IVDR poses restrictions on the use of laboratory developed tests (LDTs). To provide a uniform interpretation of the IVDR for colleagues in clinical practice, the IVDR Task Force was created by the scientific societies of laboratory specialties in the Netherlands. A guidance document with explanations and interpretations of relevant passages of the IVDR was drafted to help laboratories prepare for the impact of this new legislation. Feedback from interested parties and stakeholders was collected and used to further improve the document. Here we would like to present our approach to our European colleagues and inform them about the impact of the IVDR and, importantly we would like to present potentially useful approaches to fulfill the requirements of the IVDR for LDTs.
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http://dx.doi.org/10.1515/cclm-2020-1384DOI Listing
November 2020

Genital self-sampling compared with cervicovaginal lavage for the diagnosis of female genital schistosomiasis in Zambian women: The BILHIV study.

PLoS Negl Trop Dis 2020 07 14;14(7):e0008337. Epub 2020 Jul 14.

Department of Clinical Research, London School of Hygiene and Tropical Medicine, London, United Kingdom.

Background: Given the potentially causal association of female genital schistosomiasis (FGS) with HIV-1 infection, improved diagnostics are urgently needed to scale-up FGS surveillance. The BILHIV (bilharzia and HIV) study assessed the performance of home-based self-collection methods (cervical and vaginal swabs) compared to cervicovaginal lavage (CVL) for the detection of Schistosoma DNA by real-time polymerase chain reaction (PCR).

Methods: Between January and August 2018, a consecutive series of female participants from the Population-Cohort of the previous HIV prevention trial HPTN 071 (PopART), resident in Livingstone, Zambia were invited to take part in BILHIV if they were 18-31 years old, non-pregnant and sexually active. Genital self-collected swabs and a urine specimen were obtained and a questionnaire completed at home visits. CVL was obtained at clinic follow-up.

Results: 603 women self-collected genital swabs. Of these, 527 women had CVL performed by a mid-wife during clinic follow-up. Schistosoma DNA was more frequently detected in genital self-collected specimens (24/603, 4.0%) compared to CVL (14/527, 2.7%). Overall, 5.0% (30/603) women had female genital schistosomiasis, defined as a positive PCR by any genital sampling method (cervical swab PCR, vaginal swab PCR, or CVL PCR) and 95% (573/603) did not have a positive genital PCR. The sensitivity of any positive genital self-collected swab against CVL was 57.1% (95% CI 28.9-82.3%), specificity 97.3% (95.5-98.5%). In a subset of participants with active schistosome infection, determined by detectable urine Circulating Anodic Antigen (CAA) (15.1%, 91/601), positive PCR (4.3%, 26/601), or positive microscopy (5.5%, 33/603), the sensitivity of any positive self-collected specimen against CVL was 88.9% (51.8-99.7%).

Conclusions: Genital self-sampling increased the overall number of PCR-based FGS diagnoses in a field setting, compared with CVL. Home-based sampling may represent a scalable alternative method for FGS community-based diagnosis in endemic resource limited settings.
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http://dx.doi.org/10.1371/journal.pntd.0008337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360036PMC
July 2020

A double-blind, placebo-controlled phase 1/2a trial of the genetically attenuated malaria vaccine PfSPZ-GA1.

Sci Transl Med 2020 05;12(544)

Radboudumc Center for Infectious Diseases, Department of Medical Microbiology, Radboud University Medical Center, 6525 GA Nijmegen, Netherlands.

Immunization with attenuated sporozoites can induce protection against malaria infection, as shown by (Pf) sporozoites attenuated by radiation in multiple clinical trials. As alternative attenuation strategy with a more homogeneous population of Pf sporozoites (PfSPZ), genetically engineered sporozoites (SPZ) lacking the genes b9 and slarp induced sterile protection against malaria in mice. Consequently, PfSPZ-GA1 Vaccine, a Pf identical double knockout (Pf∆∆), was generated as a genetically attenuated malaria parasite vaccine and tested for safety, immunogenicity, and preliminary efficacy in malaria-naïve Dutch volunteers. Dose-escalation immunizations up to 9.0 × 10 PfSPZ of PfSPZ-GA1 Vaccine were well tolerated without breakthrough blood-stage infection. Subsequently, groups of volunteers were immunized three times by direct venous inoculation with cryopreserved PfSPZ-GA1 Vaccine (9.0 × 10 or 4.5 × 10 PfSPZ, = 13 each), PfSPZ Vaccine (radiation-attenuated PfSPZ, 4.5 × 10 PfSPZ, = 13), or normal saline placebo at 8-week intervals, followed by exposure to mosquito bite controlled human malaria infection (CHMI). After CHMI, 3 of 25 volunteers from both PfSPZ-GA1 groups were sterilely protected, and the remaining 17 of 22 showed a patency ≥9 days (median patency in controls, 7 days; range, 7 to 9). All volunteers in the PfSPZ Vaccine control group developed parasitemia (median patency, 9 days; range, 7 to 12). Immunized groups exhibited a significant, dose-related increase in anti-Pf circumsporozoite protein (CSP) antibodies and Pf-specific interferon-γ (IFN-γ)-producing T cells. Although no definite conclusion can be drawn on the potential strength of protective efficacy of PfSPZ-GA1 Vaccine, the favorable safety profile and induced immune responses by PfSPZ-GA1 Vaccine warrant further clinical evaluation.
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http://dx.doi.org/10.1126/scitranslmed.aaz5629DOI Listing
May 2020

Performance of the QIAstat-Dx Gastrointestinal Panel for Diagnosing Infectious Gastroenteritis.

J Clin Microbiol 2020 02 24;58(3). Epub 2020 Feb 24.

Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands

Detection and identification of enteropathogens that cause infectious gastroenteritis are essential steps for appropriate patient treatment and effective isolation precautions. Several syndrome-based tests have recently become available, with the gastrointestinal panel (GIP) assay on the QIAstat-Dx as the most recent addition to the syndromic testing landscape. The QIAstat-Dx GIP assay offers simultaneous testing for 24 bacterial, viral, and parasitic enteropathogens using a single test that reports the results in 70 min. In this study, we compared the performance of the GIP assay to laboratory-developed real-time PCR assays (LDTs), using 172 prospectively and retrospectively collected fecal samples from patients suspected to have infectious gastroenteritis. The GIP assay detected 97/107 enteropathogens (91%) that were detected by LDTs, and the overall agreement of results increased to 95% when excluding discrepant results with cycle threshold ( ) values of >35. Further, the GIP assay detected 42 additional enteropathogens that were not detected, or tested, by LDTs. These included 35 diarrheagenic targets for which the clinical relevance is unclear for most. The main advantage of the QIAstat-Dx system compared to other syndromic testing systems is the ability to generate values that could help with the interpretation of results. However, compared to LDTs, the GIP assay is limited by flexibility and high-throughput testing. In conclusion, the GIP assay offers an easy, sample-to-answer workflow with a rapid detection of the most common enteropathogens and therefore has the potential to direct appropriate therapy and infection control precautions.
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http://dx.doi.org/10.1128/JCM.01737-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041566PMC
February 2020

Clinical implications of rapid ePlex® Respiratory Pathogen Panel testing compared to laboratory-developed real-time PCR.

Eur J Clin Microbiol Infect Dis 2018 Mar 8;37(3):571-577. Epub 2017 Dec 8.

Department of Medical Microbiology, Leiden University Medical Center, P.O. Box 9600, 2300 RC, Leiden, The Netherlands.

Rapid diagnosis of respiratory infections is of great importance for adequate isolation and treatment. Due to the batch-wise testing, laboratory-developed real-time polymerase chain reaction (PCR) assays (LDT) often result in a time to result of one day. Here, LDT was compared with rapid ePlex® Respiratory Pathogen (RP) Panel testing of GenMark Diagnostics (Carlsbad, CA, USA) with regard to time to result, installed isolation precautions, and antibacterial/antiviral treatment. Between January and March 2017, 68 specimens of 64 patients suspected of an acute respiratory infection were tested with LDT and the ePlex® RP panel. The time to result was calculated as the time between sample reception and result reporting. Information regarding isolation and antibacterial/antiviral treatment was obtained from the patient records. Thirty specimens tested LDT positive (47%) and 29 ePlex® RP panel positive (45%). The median time to result was 27.1 h (range 6.5-96.6) for LDT versus 3.4 h (range 1.5-23.6) for the RP panel, p-value < 0.001. In 14 out of 30 patients, isolation was discontinued based on the ePlex® RP panel results, saving 21 isolation days. ePlex® RP panel test results were available approximately one day ahead of the LDT results in the 19 patients receiving antiviral/antibacterial treatment. In addition, two bacterial pathogens, not requested by the physician, were detected using the RP panel. Analysis of respiratory infections with the ePlex® RP panel resulted in a significant decrease in time to result, enabling a reduction in isolation days in half of the patients. Furthermore, syndromic RP panel testing increased the identification of causative pathogens.
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http://dx.doi.org/10.1007/s10096-017-3151-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816761PMC
March 2018

T and B Cell Markers in Dried Blood Spots of Neonates with Congenital Cytomegalovirus Infection: B Cell Numbers at Birth Are Associated with Long-Term Outcomes.

J Immunol 2017 01 30;198(1):102-109. Epub 2016 Nov 30.

Department of Medical Microbiology, Leiden University Medical Center, 2333 ZA Leiden, the Netherlands.

Congenital CMV infection (cCMV) is the most common congenital infection that can cause long-term impairment (LTI). The pathogenesis of LTI is not completely understood. Fetal immunity may play a role in controlling the infection and preventing LTI, although immune activation may also contribute to fetal immunopathology. In this study, we analyzed various molecular markers of T and B cell numbers in neonatal dried blood spots of 99 children with cCMV and 54 children without cCMV: δRec-ψJα signal joints on TCR excision circles, intron recombination signal sequence k-deleting element signal joints on Igκ-deleting recombination excision circles, genomic intron recombination signal sequence k-deleting element coding joint, genomic Vδ1-Jδ1, and Vδ2-Jδ1 rearrangements. Of this cohort, clinical symptoms at birth and LTI at 6 y of age were recorded. Neonates with cCMV had fewer TCR excision circles in their blood than non-infected controls. Furthermore, cCMV infection was associated with increased numbers of γδ T cells and B cells, and these numbers were positively correlated with CMV viral load in the dried blood spots. Infected children with a better long-term outcome had higher numbers of B cells at birth than those who developed LTI; no difference in B cell replication was observed. The potential protective role of B cells in controlling cCMV-related disease and the clinical value of this marker as a predictor of long-term outcome merit further evaluation.
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http://dx.doi.org/10.4049/jimmunol.1601182DOI Listing
January 2017

MALDI-TOF mass spectrometry for differentiation between Streptococcus pneumoniae and Streptococcus pseudopneumoniae.

Diagn Microbiol Infect Dis 2016 May 22;85(1):9-11. Epub 2016 Jan 22.

Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.

We compared the Vitek MS and Microflex MALDI-TOF mass spectrometry platform for species differentiation within the Streptococcus mitis group with PCR assays targeted at lytA, Spn9802, and recA as reference standard. The Vitek MS correctly identified 10/11 Streptococcus pneumoniae, 13/13 Streptococcus pseudopneumoniae, and 12/13 S. mitis/oralis. The Microflex correctly identified 9/11 S. pneumoniae, 0/13 S. pseudopneumoniae, and 13/13 S. mitis/oralis. MALDI-TOF is a powerful tool for species determination within the mitis group. Diagnostic accuracy varies depending on platform and database used.
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http://dx.doi.org/10.1016/j.diagmicrobio.2016.01.012DOI Listing
May 2016

Increased expression levels of chromosomal AmpC β-lactamase in clinical Escherichia coli isolates and their effect on susceptibility to extended-spectrum cephalosporins.

Microb Drug Resist 2015 Feb 4;21(1):7-16. Epub 2014 Sep 4.

1 Department of Medical Microbiology, Leiden University Medical Center , Leiden, The Netherlands .

Forty-nine clinical Escherichia coli isolates, both extended-spectrum β-lactamase (ESBL) negative and ESBL positive, were studied to investigate whether increased AmpC expression is a mechanism involved in cefoxitin resistance and if this influences the third-generation cephalosporin activity. Nine of 33 (27.2%) cefoxitin-resistant (minimum inhibitory concentration [MIC] >8 mg/L) isolates showed hyperproduction of chromosomal AmpC (c-AmpC) based on (1) at least two positive tests using AmpC inhibitors, (2) mutations in the promoter/attenuator regions, and (3) a 6.1- to 163-fold increase in c-ampC expression by quantitative reverse transcription-polymerase chain reaction. In ESBL-negative isolates, MICs of ceftazidime and cefotaxime were mostly above the wild-type (WT) level, but below the S/I breakpoint (EUCAST guideline), except for one isolate with MICs of 4 mg/L. No plasmid-mediated AmpCs were found. Periplasmic extracts of nine c-AmpC hyperproducers were preincubated with or without cefuroxime or ceftazidime and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cefuroxime and ceftazidime were stable to hydrolysis but acted as inhibitors of the enzyme. None of these isolates showed loss of porins. Thus, cefoxitin resistance has low specificity for detecting upregulated c-AmpC production. c-AmpC hyperproducing E. coli is mostly still susceptible to third-generation cephalosporins but less than WT E. coli. Surveillance of cefoxitin-resistant E. coli to monitor developments in the activity of third-generation cephalosporins against c-AmpC hyperproducers is warranted.
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http://dx.doi.org/10.1089/mdr.2014.0108DOI Listing
February 2015

Cytomegalovirus DNA detection in dried blood spots and perilymphatic fluids from pediatric and adult cochlear implant recipients with prelingual deafness.

J Clin Virol 2013 Feb 9;56(2):113-7. Epub 2012 Nov 9.

Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.

Background: Congenital cytomegalovirus (CMV) infection is the leading cause of non-genetic congenital hearing loss. The contribution of congenital CMV to prelingual deafness and the pathophysiology is largely unknown.

Objective: (1) To analyze the prevalence of congenital CMV among cochlear implant (CI) recipients with prelingual deafness. (2) To genotype CMV present in dried blood spots (DBS) and in the inner ear years after birth.

Study Design: Children and adults with prelingual deafness who received a CI in 2010-2011 were included prospectively. Perilymphatic fluids were collected during CI surgery and, in the pediatric cases, DBS were retrieved for CMV DNA detection. Furthermore, a cohort of children with prelingual deafness who received a CI between 2003 and 2008 were included retrospectively. CMV detection in DBS and perilymph was followed by gB and gH genotyping.

Results: Seventysix pediatric CI recipients were included. Seventy DBS were tested for CMV DNA, resulting in a prevalence of congenital CMV of 14% (10/70). Perilymphatic fluid was available from 29 pediatric CI recipients. One perilymph fluid, of a 21-month old girl with congenital CMV, asymptomatic at birth, was CMV DNA positive. The CMV strain in the perilymph was genotypically identical to the strain present in her DBS (gB1/gH2). Perilymph samples from 21 adult CI recipients were CMV DNA negative.

Conclusions: Our study stresses the important contribution of congenital CMV among pediatric CI recipients. Furthermore, our genotyping data support the hypothesis that CMV-related hearing loss is associated with ongoing viral replication in the inner ear up to years after birth.
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http://dx.doi.org/10.1016/j.jcv.2012.10.008DOI Listing
February 2013

Evaluation of several biochemical and molecular techniques for identification of Streptococcus pneumoniae and Streptococcus pseudopneumoniae and their detection in respiratory samples.

J Clin Microbiol 2012 Apr 25;50(4):1171-7. Epub 2012 Jan 25.

Department of Medical Microbiology, Leiden University Medical Center, Leiden, Netherlands.

The identification and detection of mitis group streptococci, which contain Streptococcus pneumoniae, have been hampered by the lack of sensitive and specific assays. In this study, we evaluated several biochemical and molecular assays for the identification of S. pneumoniae and Streptococcus pseudopneumoniae and their distinction from other mitis group streptococci using a collection of 54 isolates obtained by the routine culturing of 53 respiratory specimens from patients with community-acquired pneumonia. The combined results of the biochemical and molecular assays indicated the presence of 23 S. pneumoniae, 2 S. pseudopneumoniae, and 29 other mitis group streptococcal isolates. The tube bile solubility test that is considered gold standard for the identification of S. pneumoniae showed concordant results with optochin susceptibility testing (CO(2) atmosphere) and a real-time multiplex PCR assay targeting the Spn9802 fragment and the autolysin gene. Optochin susceptibility testing upon incubation in an O(2) atmosphere, bile solubility testing by oxgall disk, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and sequence analysis of the tuf and rpoB genes resulted in several false-positive, false-negative, or inconclusive results. The S. pseudopneumoniae isolates could be identified only by molecular assays, and the multiplex real-time PCR assay was concluded to be most convenient for the identification of S. pneumoniae and S. pseudopneumoniae isolates. Using this method, S. pneumoniae and S. pseudopneumoniae DNA could be detected in the respiratory samples from which they were isolated and in an additional 11 samples from which only other streptococci were isolated.
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http://dx.doi.org/10.1128/JCM.06609-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318541PMC
April 2012

Rapid genotyping of cytomegalovirus in dried blood spots by multiplex real-time PCR assays targeting the envelope glycoprotein gB and gH genes.

J Clin Microbiol 2012 Feb 23;50(2):232-7. Epub 2011 Nov 23.

Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.

Genotyping of cytomegalovirus (CMV) is useful to examine potential differences in the pathogenicity of strains and to demonstrate coinfection with multiple strains involved in CMV disease in adults and congenitally infected newborns. Studies on genotyping of CMV in dried blood spots (DBS) are rare and have been hampered by the small amount of dried blood available. In this study, two multiplex real-time PCR assays for rapid gB and gH genotyping of CMV in DBS were developed. Validation of the assays with 39 CMV-positive plasma samples of transplant recipients and 21 urine specimens of congenitally infected newborns was successful in genotyping 100% of the samples, with gB1 and gB3 being the most prevalent genotypes. Multiple gB and gH genotypes were detected in 36% and 33% of the plasma samples, respectively. One urine sample from a newborn with symptomatic congenital CMV was positive for gB1 and gB2. DBS of congenitally infected newborns (n = 41) were tested using 9 μl of dried blood, and genotypes were detected in 81% (gB) and 73% (gH) of the samples, with gB3 being the most prevalent genotype. No clear association of specific genotypes with clinical outcome was observed. In conclusion, the CMV gB and gH PCR assays were found to be rapid, sensitive for detecting mixed infections, and suitable for direct usage on DBS. These assays are efficient tools for genotyping of CMV in DBS of congenitally infected newborns.
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http://dx.doi.org/10.1128/JCM.05253-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3264199PMC
February 2012

[Outbreak of Shiga toxin-producing Escherichia coli and haemolytic uraemic syndrome].

Ned Tijdschr Geneeskd 2011 ;155(35):A3743

Leids Universitair Medisch Centrum, Centrum van Infectieziekten, Leiden, the Netherlands.

Enterohaemorrhagic Escherichia coli (EHEC) is a group of pathogenic Shiga toxin-producing E. coli that can cause haemorrhagic colitis and haemolytic uraemic syndrome (HUS). The disease usually occurs sporadically, but sometimes also occurs in large outbreaks such as that which recently occurred in northern Germany. EHEC infection is a zoonosis and its reservoir is in ruminant farm animals (cattle, sheep and goats). EHEC infection should be considered in patients with bloody diarrhoea, but in the course of many severe EHEC infections a picture resembling HUS may also occur. Antibiotic treatment is contraindicated because it does not reduce the duration of the disease and may have negative complications. Patients with EHEC infection may spread the bacteria and their care includes contact isolation measures with their own toilet facilities. The E. coli type O104:H4 that occurred in Germany has a combination of specific virulence characteristics. This outbreak affected many people who developed HUS and neurological symptoms following bloody diarrhoea.
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November 2011

First report of Atopobium vaginae bacteremia with fetal loss after chorionic villus sampling.

J Clin Microbiol 2011 Apr 2;49(4):1684-6. Epub 2011 Feb 2.

Department of Medical Microbiology, Leiden University Medical Center, Postbus 9600, 2300 RC Leiden, The Netherlands.

Infectious complications after chorionic villus sampling (CVS) are rare (<0.1%) but can lead to maternal sepsis and spontaneous abortion. We report the first bacteremia with Atopobium vaginae and suggest A. vaginae to be a pathogenic microorganism that can lead to intrauterine infection and fetal death following CVS.
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http://dx.doi.org/10.1128/JCM.01655-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3122803PMC
April 2011

Diagnosis of viral gastroenteritis by simultaneous detection of Adenovirus group F, Astrovirus, Rotavirus group A, Norovirus genogroups I and II, and Sapovirus in two internally controlled multiplex real-time PCR assays.

J Clin Virol 2010 Nov 9;49(3):205-10. Epub 2010 Sep 9.

Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.

Background: Norovirus, Rotavirus group A, Astrovirus, Sapovirus and Adenovirus serotypes 40 and 41, are common causes of gastroenteritis. Conventional diagnosis of these causative agents is based on antigen detection and electron microscopy.

Objective: To improve the diagnostic possibilities for viral gastroenteritis, two internally controlled multiplex real-time PCRs have been developed.

Study Design: Individual real-time PCRs were developed and optimized for the specific detection of Norovirus genogroup I, Norovirus genogroup II, Rotavirus group A, Astrovirus, Adenovirus group F and Sapovirus. Subsequently, the PCRs were combined to two multiplex PCR reactions. The multiplex assays were clinically evaluated using 239 fecal samples submitted to our laboratory over a 1-year period for the routine detection of Rotavirus and/or Adenovirus antigens using the Vikia(®) Rota/Adeno test (bioMérieux, Boxtel, The Netherlands).

Results: In general, the multiplex real-time PCR assays showed comparable sensitivity and specificity to the individual assays. A retrospective clinical evaluation showed increased pathogen detection in samples from 14% using conventional methods to 45% using PCR. Subsequently, the assay was implemented as a routine diagnostic tool. From September 2007 up to December 2009, 486 positive results were obtained in 1570 samples (31%) analyzed. Norovirus genogroup II was found the most frequently (61.1%), followed by Adenovirus (9.9%), Rotavirus (9.3%), Astrovirus (6.0%), Norovirus genogroup I (3.3%) and Sapovirus (0.4%).

Conclusions: Two internally controlled multiplex real-time PCR assays for the simultaneous detection of Astrovirus, Adenovirus group F, Rotavirus, Norovirus genogroups I and II and Sapovirus have shown significant improvement in the diagnosis of viral gastroenteritis.
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http://dx.doi.org/10.1016/j.jcv.2010.07.019DOI Listing
November 2010

Differential membrane association properties and regulation of class I and class II Arfs.

Traffic 2009 Mar 5;10(3):316-23. Epub 2008 Jan 5.

Department of Medical Microbiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

ADP-ribosylation factor (Arf) proteins are small guanosine triphosphatases (GTPases) that act as major regulators of intracellular vesicular trafficking and secretory organelle pathway integrity. Like all small monomeric GTPases, Arf proteins cycle between a GDP-bound and a GTP-bound state, and this cycling is catalysed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins. While the class I Arfs, especially Arf1, have been studied extensively, little is known as yet about the function and regulation of class II Arfs, Arf4 and Arf5. In this study, we show that Arf proteins show class-specific dynamic behaviour. Moreover, unlike class I Arfs, membrane association of class II Arfs is resistant to inhibition of large Arf GEFs by Brefeldin A. Through the construction of Arf chimeric proteins, evidence is provided that the N-terminal amphipathic helix and a class-specific residue in the conserved interswitch domain determine the membrane-binding properties of class I and class II Arf proteins. Our results show that fundamental differences exist in behaviour and regulation of these small GTPases.
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http://dx.doi.org/10.1111/j.1600-0854.2008.00868.xDOI Listing
March 2009

Reduction of phospholipase D activity during coxsackievirus infection.

J Gen Virol 2007 Nov;88(Pt 11):3027-3030

Department of Medical Microbiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands.

During enterovirus infection, host cell membranes are rigorously rearranged and modified. One ubiquitously expressed lipid-modifying enzyme that might contribute to these alterations is phospholipase D (PLD). Here, we investigated PLD activity in coxsackievirus-infected cells. We show that PLD activity is not required for efficient coxsackievirus RNA replication. Instead, PLD activity rapidly decreased upon infection and upon ectopic expression of the viral 3A protein, which inhibits the PLD activator ADP-ribosylation factor 1. However, similar decreases were observed during infection with coxsackieviruses carrying defective mutant 3A proteins. Possible causes for the reduction of PLD activity and the biological consequences are discussed.
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http://dx.doi.org/10.1099/vir.0.83169-0DOI Listing
November 2007

Impact of live cell imaging on coated vesicle research.

Semin Cell Dev Biol 2007 Aug 7;18(4):412-23. Epub 2007 Jul 7.

Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany.

The role of membrane traffic is to transfer cargo between distinct subcellular compartments. Each individual trafficking event involves the creation, transport and fusion of vesicular and tubular carriers that are formed and regulated via cytoplasmic coat protein complexes. The dynamic nature of this process is therefore highly suitable for studying using live cell imaging techniques. Although these approaches have raised further questions for the field, they have also been instrumental in providing essential new information, in particular relating to the morphology of transport carriers and the exchange kinetics of coat proteins and their regulators on membranes. Here, we present an overview of live cell-imaging experiments that have been used in the study of coated-vesicle transport, and provide specific examples of their impact on our understanding of coat function.
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http://dx.doi.org/10.1016/j.semcdb.2007.07.002DOI Listing
August 2007

Molecular determinants of the interaction between coxsackievirus protein 3A and guanine nucleotide exchange factor GBF1.

J Virol 2007 May 28;81(10):5238-45. Epub 2007 Feb 28.

Department of Medical Microbiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands.

The 3A protein of coxsackievirus B3 (CVB3), a small membrane protein that forms homodimers, inhibits endoplasmic reticulum-to-Golgi complex transport. Recently, we described the underlying mechanism by showing that the CVB3 3A protein binds to and inhibits the function of GBF1, a guanine nucleotide exchange factor for ADP-ribosylation factor 1 (Arf1), thereby interfering with Arf1-mediated COP-I recruitment. This study was undertaken to gain more insight into the molecular determinants underlying the interaction between 3A and GBF1. Here we show that 3A mutants that have lost the ability to dimerize are no longer able to bind to GBF1 and trap it on membranes. Moreover, we identify a conserved region in the N terminus of 3A that is crucial for GBF1 binding but not for 3A dimerization. Analysis of the binding domain in GBF1 showed that the extreme N terminus, the dimerization/cyclophilin binding domain, and the homology upstream of Sec7 domain are required for the interaction with 3A. In contrast to that of full-length GBF1, overexpression of a GBF1 mutant lacking its extreme N terminus failed to rescue the effects of 3A. Together, these data provide insight into the molecular requirements of the interaction between 3A and GBF1.
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http://dx.doi.org/10.1128/JVI.02680-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1900206PMC
May 2007

Effects of picornavirus 3A Proteins on Protein Transport and GBF1-dependent COP-I recruitment.

J Virol 2006 Dec 27;80(23):11852-60. Epub 2006 Sep 27.

Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.

The 3A protein of the coxsackievirus B3 (CVB3), an enterovirus that belongs to the family of the picornaviruses, inhibits endoplasmic reticulum-to-Golgi transport. Recently, we elucidated the underlying mechanism by showing that CVB3 3A interferes with ADP-ribosylation factor 1 (Arf1)-dependent COP-I recruitment to membranes by binding and inhibiting the function of GBF1, a guanine nucleotide exchange factor that is required for the activation of Arf1 (E. Wessels et al., Dev. Cell 11:191-201, 2006). Here, we show that the 3A protein of poliovirus, another enterovirus, is also able to interfere with COP-I recruitment through the same mechanism. No interference with protein transport or COP-I recruitment was observed for the 3A proteins of any of the other picornaviruses tested here (human rhinovirus [HRV], encephalomyocarditis virus, foot-and-mouth disease virus, and hepatitis A virus). We show that the 3A proteins of HRV, which are the most closely related to the enteroviruses, are unable to inhibit COP-I recruitment, due to a reduced ability to bind GBF1. When the N-terminal residues of the HRV 3A proteins are replaced by those of CVB3 3A, chimeric proteins are produced that have gained the ability to bind GBF1 and, by consequence, to inhibit protein transport. These results show that the N terminus of the CVB3 3A protein is important for binding of GBF1 and its transport-inhibiting function. Taken together, our data demonstrate that the activity of the enterovirus 3A protein to inhibit GBF1-dependent COP-I recruitment is unique among the picornaviruses.
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http://dx.doi.org/10.1128/JVI.01225-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1642585PMC
December 2006

A viral protein that blocks Arf1-mediated COP-I assembly by inhibiting the guanine nucleotide exchange factor GBF1.

Dev Cell 2006 Aug;11(2):191-201

Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, PO Box 9101, 6500 HB Nijmegen, The Netherlands.

Many viruses modify cellular processes for their own benefit. The enterovirus 3A protein inhibits endoplasmic reticulum (ER)-to-Golgi transport, a function previously suggested to be important for viral suppression of immune responses. Here, we show that a virus carrying a 3A protein defective in inhibiting ER-to-Golgi transport is indeed less virulent in mice, and we unravel the mechanism by which 3A inhibits this trafficking step. Evidence is provided that 3A inhibits the activation of the GTPase ADP-ribosylation factor 1 (Arf1), which regulates the recruitment of the COP-I coat complex to membranes. 3A specifically inhibits the function of GBF1, a guanine nucleotide exchange factor for Arf1, by interacting with its N terminus. By specifically interfering with GBF1-mediated Arf1 activation, 3A may prove a valuable tool in dissecting the early steps of the secretory pathway.
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http://dx.doi.org/10.1016/j.devcel.2006.06.005DOI Listing
August 2006

Structure-function analysis of the coxsackievirus protein 3A: identification of residues important for dimerization, viral rna replication, and transport inhibition.

J Biol Chem 2006 Sep 25;281(38):28232-43. Epub 2006 Jul 25.

Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, 6500 HB Nijmegen, The Netherlands.

The coxsackievirus B3 3A protein forms homodimers and plays important roles in both viral RNA (vRNA) replication and the viral inhibition of intracellular protein transport. The molecular determinants that are required for each of these functions are yet poorly understood. Based on the NMR structure of the closely related poliovirus 3A protein, a molecular model of the coxsackievirus B3 3A protein was constructed. Using this structural model, specific mutants were designed to study the structure-function relationship of 3A. The mutants were tested for their capacity to dimerize, support vRNA replication, and block protein transport. A hydrophobic interaction between the monomers and an intermolecular salt bridge were identified as major determinants required for dimerization. We show that dimerization is important for both efficient vRNA replication and inhibition of protein transport. In addition, determinants were identified that were not required for dimerization but that were essential for either one of the biological functions of 3A. The combination of the in silico and in vivo results obtained in this study provides important insights in both the structural and functional aspects of 3A.
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http://dx.doi.org/10.1074/jbc.M601122200DOI Listing
September 2006

A proline-rich region in the coxsackievirus 3A protein is required for the protein to inhibit endoplasmic reticulum-to-golgi transport.

J Virol 2005 Apr;79(8):5163-73

Department of Medical Microbiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.

The ability of the 3A protein of coxsackievirus B (CVB) to inhibit protein secretion was investigated for this study. Here we show that the ectopic expression of CVB 3A blocked the transport of both the glycoprotein of vesicular stomatitis virus, a membrane-bound secretory marker, and the alpha-1 protease inhibitor, a luminal secretory protein, at a step between the endoplasmic reticulum (ER) and the Golgi complex. CVB 3A contains a conserved proline-rich region in its N terminus. The importance of this proline-rich region was investigated by introducing Pro-to-Ala substitutions. The mutation of Pro19 completely abolished the ability of 3A to inhibit ER-to-Golgi transport. The mutation of Pro14, Pro17, or Pro20 also impaired this ability, but to a lesser extent. The mutation of Pro18 had no effect. We also investigated the possible importance of this proline-rich region for the function of 3A in viral RNA replication. To this end, we introduced the Pro-to-Ala mutations into an infectious cDNA clone of CVB3. The transfection of cells with in vitro-transcribed RNAs of these clones gave rise to mutant viruses that replicated with wild-type characteristics. We concluded that the proline-rich region in CVB 3A is required for its ability to inhibit ER-to-Golgi transport, but not for its function in viral RNA replication. The functional relevance of the proline-rich region is discussed in light of the proposed structural model of 3A.
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http://dx.doi.org/10.1128/JVI.79.8.5163-5173.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1069528PMC
April 2005

Determinants for membrane association and permeabilization of the coxsackievirus 2B protein and the identification of the Golgi complex as the target organelle.

J Biol Chem 2003 Jan 18;278(2):1012-21. Epub 2002 Sep 18.

Department of Medical Microbiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, P. O. Box 9100, The Netherlands.

The 2B protein of enterovirus is responsible for the alterations in the permeability of secretory membranes and the plasma membrane in infected cells. The structural requirements for the membrane association and the subcellular localization of this essential virus protein, however, have not been defined. Here, we provide evidence that the 2B protein is an integral membrane protein in vivo that is predominantly localized at the Golgi complex upon individual expression. Addition of organelle-specific targeting signals to the 2B protein revealed that the Golgi localization is an absolute prerequisite for the ability of the protein to modify plasma membrane permeability. Expression of deletion mutants and heterologous proteins containing specific domains of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in the Golgi complex and the possible mechanism by which a Golgi-localized virus protein modifies plasma membrane permeability are discussed.
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http://dx.doi.org/10.1074/jbc.M207745200DOI Listing
January 2003