Laboratory for Medical Microbiology and Immunology

Velp, Netherlands

Laboratory for Medical Microbiology and Immunology

Velp, Netherlands
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Wassenberg M.W.M.,University Utrecht | Kluytmans J.A.J.W.,Amphia Hospital | Kluytmans J.A.J.W.,VU University Amsterdam | Box A.T.A.,University Utrecht | And 17 more authors.
Clinical Microbiology and Infection | Year: 2010

Pre-emptive isolation of suspected methicillin-resistant Staphylococcus aureus (MRSA) carriers is considered essential for controlling the spread of MRSA, but noncolonized patients will be isolated unnecessarily as a result of a delay in diagnosis of 3-5 days with conventional cultures. We determined costs per isolation day avoided, and incremental costs of rapid MRSA screening tests when added to conventional screening, but with decisions on isolation measures based on PCR results. A prospective multicentre study evaluating BD GeneOhm MRSA PCR ('IDI') (BD Diagnostics, San Diego, CA, USA), Xpert MRSA ('GeneXpert') (Cepheid, Sunnyvale, CA, USA) and chromogenic agar (MRSA-ID) (bioMérieux, Marcy-l'Etoile, France) was performed in 14 Dutch hospitals. Among 1764 patients at risk, MRSA prevalence was 3.3% (n = 59). Duration of isolation was 19.7 and 16.1 h with IDI and GeneXpert, respectively, and would have been 30.0 and 76.2 h when based on chromogenic agar and conventional cultures, respectively. Negative predictive values (at a patient level) were 99.5%, 99.1% and 99.5% for IDI, GeneXpert and chromogenic agar, respectively. Numbers of isolation days were reduced by 60% and 47% with PCR-based and chromogenic agar-based screening, respectively. The cost per test was €56.22 for IDI, €69.62 for GeneXpert and €2.08 for chromogenic agar, and additional costs per extra isolation day were €26.34. Costs per isolation day avoided were €95.77 (IDI) and €125.43 (GeneXpert). PCR-based decision-making added €153.64 (IDI) and €193.84 (GeneXpert) per patient to overall costs and chromogenic testing would have saved €30.79 per patient. Rapid diagnostic testing safely reduces the number of unnecessary isolation days, but only chromogenic screening, and not PCR-based screening, can be considered as cost saving. © 2010 The Authors. Journal Compilation © 2010 European Society of Clinical Microbiology and Infectious Diseases.


Wassenberg M.,University Utrecht | Kluytmans J.,Amphia Hospital | Kluytmans J.,VU University Amsterdam | Erdkamp S.,University Utrecht | And 16 more authors.
Critical Care | Year: 2012

Introduction: Pre-emptive isolation of suspected methicillin-resistant Staphylococcus aureus (MRSA) carriers is a cornerstone of successful MRSA control policies. Implementation of such strategies is hampered when using conventional cultures with diagnostic delays of three to five days, as many non-carriers remain unnecessarily isolated. Rapid diagnostic testing (RDT) reduces the amount of unnecessary isolation days, but costs and benefits have not been accurately determined in intensive care units (ICUs).Methods: Embedded in a multi-center hospital-wide study in 12 Dutch hospitals we quantified cost per isolation day avoided using RDT for MRSA, added to conventional cultures, in ICUs. BD GeneOhm™ MRSA PCR (IDI) and Xpert MRSA (GeneXpert) were subsequently used during 17 and 14 months, and their test characteristics were calculated with conventional culture results as reference. We calculated the number of pre-emptive isolation days avoided and incremental costs of adding RDT.Results: A total of 163 patients at risk for MRSA carriage were screened and MRSA prevalence was 3.1% (n = 5). Duration of isolation was 27.6 and 21.4 hours with IDI and GeneXpert, respectively, and would have been 96.0 hours when based on conventional cultures. The negative predictive value was 100% for both tests. Numbers of isolation days were reduced by 44.3% with PCR-based screening at the additional costs of €327.84 (IDI) and €252.14 (GeneXpert) per patient screened. Costs per isolation day avoided were €136.04 (IDI) and €121.76 (GeneXpert).Conclusions: In a low endemic setting for MRSA, RDT safely reduced the number of unnecessary isolation days on ICUs by 44%, at the costs of €121.76 to €136.04 per isolation day avoided. © 2012 Wassenberg et al.; licensee BioMed Central Ltd.


Nijhuis R.H.T.,Laboratory for Medical Microbiology and Immunology | Oueslati S.,University Paris - Sud | Zhou K.,University of Groningen | Bosboom R.W.,Laboratory for Medical Microbiology and Immunology | And 2 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2014

Objectives: Klebsiella oxytoca is a member of the family of Enterobacteriaceae and often contains the β-lactamase blaOXY gene. Although this β-lactamase does not naturally hydrolyse ceftazidime, this study describes possible in vivo selection of a clinical K. oxytoca isolate showing increased MICs of ceftazidime. Methods: To reveal the molecular mechanism underlying this unusual resistance phenotype, WGS, cloning, overexpression, MIC and steady-state kinetic studies were performed. Results: A patient was treated for a septic episode with ceftazidime (4 g/day). This therapy was based on earlier culture results in which, amongst others, a K. oxytoca (Velp-1) isolate was identified. After 11 days of treatment, K. oxytoca Velp-2 was isolated from a pus sample drained from the wound. The isolate showed increased resistance to ceftazidime (MIC ≥64 mg/L) compared with the original K. oxytoca isolate (Velp-1). WGS revealed the presence of a novel blaOXY-2 allele, designated blaOXY-2-15, with a two amino acid deletion at Ambler positions 168 and 169 compared with OXY-2-2. Cloning blaOXY-2-15 into Escherichia coli TOP10 resulted in increased MICs of ceftazidime, but reduced MICs of most other b-lactams compared with OXY-2-2. Steady-state kinetics confirmed the results of the MIC data, showing clearly significant ceftazidime hydrolysis. Conclusions: This report shows the risk of in vivo selection of ceftazidime-resistant K. oxytoca isolates after prolonged ceftazidime treatment. Furthermore, it is the first known report of a K. oxytoca isolate conferring resistance to ceftazidime by a two amino acid deletion in the omega loop of OXY-2-2. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.


Wassenberg M.W.M.,University Utrecht | Kluytmans J.A.J.W.,Amphia Hospital | Kluytmans J.A.J.W.,VU University Amsterdam | Bosboom R.W.,Slingeland Hospital | And 16 more authors.
Clinical Microbiology and Infection | Year: 2011

Multiple body site screening and pre-emptive isolation of patients at risk for methicillin-resistant Staphylococcus aureus (MRSA) carriage are considered essential for control of nosocomial spread. The relative importance of extranasal screening when using rapid diagnostic testing (RDT) is unknown. Using data from a multicentre study evaluating BD GeneOhm™ MRSA PCR (IDI), Xpert MRSA (GeneXpert) and chromogenic agar, added to conventional cultures, we determined cost-effectiveness assuming isolation measures would have been based on RDT results of different hypothetical screening regimes. Costs per isolation day avoided were calculated for regimes with single or less extensive multiple site RDT, regimes without conventional back-up cultures and when PCR would have been performed with pooling of swabs. Among 1764 patients at risk, MRSA prevalence was 3.3% (n=59). In all scenarios the negative predictive value is above 98.4%. With back-up cultures of all sites as a reference, the costs per isolation day avoided were €15.19, €30.83 and €45.37 with 'nares only' screening using chromogenic agar, IDI and GeneXpert, respectively, as compared with €19.95, €95.77 and €125.43 per isolation day avoided when all body sites had been screened. Without back-up cultures costs per isolation day avoided using chromogenic agar would range from €9.24 to €76.18 when costs per false-negative RDT range from €5000 up to €50000; costs for molecular screening methods would be higher in all scenarios evaluated. In conclusion, in a low endemic setting chromogenic agar screening added to multiple site conventional cultures is the most cost-effective MRSA screening strategy. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.


Nijhuis R.H.T.,Laboratory for Medical Microbiology and Immunology | Severs T.T.,University Utrecht | van der Vegt D.S.J.M.,Laboratory for Medical Microbiology and Immunology | van der Vegt D.S.J.M.,Laboratory of Medical Microbiology | And 2 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2015

Objectives: Mycoplasma genitalium is a sexually transmitted pathogen, and infection with it is usually treated with macrolides. Unfortunately, emerging resistance to the macrolides has been associated with mutations in region V of the 23S rRNA gene. The aim of this retrospective study was to describe the incidence of macrolide resistance-associated mutations in M. genitalium from patients in the Netherlands. Methods: All urogenital samples obtained from patients visiting a general practitioner or hospital in the east of the Netherlands that tested positive using the routine M. genitalium real-time PCR (February 2012-November 2014) were included. Following a PCR targeting the 23S rRNA gene, sequencing of the PCR fragments was performed to identify possible macrolide resistance-associated mutations. Results: Forty-eight of the 153 samples (31.4%) included in this study contained a mutation in the 23S rRNA gene. This was reduced to 44/146 (30.1%) if only samples from unique patients were included. The predominant mutations identified were A2058G (16/44; 36.3%), A2059G (14/44; 31.8%) and a unique high proportion of A2058T (12/44; 27.3%). Treatment failure was observed in four patients initially infected with M. genitalium containing macrolide resistance-associated mutations, while in one of these patients subsequent treatment with moxifloxacin resulted in a microbiological cure. Conclusions: This study shows that macrolide resistance-associated mutations in M. genitalium occur with a high frequency. In contrast to studies from other regions, Dutch M. genitalium isolates carry the A2058T mutation at high frequency. Our data confirm the need for prospective detection of macrolide resistance-associated mutations prior to treating patients. © The Author 2015.


PubMed | Laboratory for Medical Microbiology and Immunology and University Utrecht
Type: Journal Article | Journal: The Journal of antimicrobial chemotherapy | Year: 2015

Mycoplasma genitalium is a sexually transmitted pathogen, and infection with it is usually treated with macrolides. Unfortunately, emerging resistance to the macrolides has been associated with mutations in region V of the 23S rRNA gene. The aim of this retrospective study was to describe the incidence of macrolide resistance-associated mutations in M. genitalium from patients in the Netherlands.All urogenital samples obtained from patients visiting a general practitioner or hospital in the east of the Netherlands that tested positive using the routine M. genitalium real-time PCR (February 2012-November 2014) were included. Following a PCR targeting the 23S rRNA gene, sequencing of the PCR fragments was performed to identify possible macrolide resistance-associated mutations.Forty-eight of the 153 samples (31.4%) included in this study contained a mutation in the 23S rRNA gene. This was reduced to 44/146 (30.1%) if only samples from unique patients were included. The predominant mutations identified were A2058G (16/44; 36.3%), A2059G (14/44; 31.8%) and a unique high proportion of A2058T (12/44; 27.3%). Treatment failure was observed in four patients initially infected with M. genitalium containing macrolide resistance-associated mutations, while in one of these patients subsequent treatment with moxifloxacin resulted in a microbiological cure.This study shows that macrolide resistance-associated mutations in M. genitalium occur with a high frequency. In contrast to studies from other regions, Dutch M. genitalium isolates carry the A2058T mutation at high frequency. Our data confirm the need for prospective detection of macrolide resistance-associated mutations prior to treating patients.

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