Sequence-based epidemiology of an OXA-48 plasmid during a hospital outbreak.

A large OXA-48 outbreak in the Netherlands involved the spread of OXA-48producing Enterobacteriaceae among at least 118 patients, suggesting horizontal transfer of this resistance gene through one or more plasmids. Elucidating transmission dynamics of resistance plasmids is hampered by the low resolution of classic typing methods. This study aimed to investigate the molecular epidemiology of plasmids carrying OXA-48 carbapenemase using a next-generation sequencing approach.

Spread of Carbapenem Resistance by Transposition and Conjugation Among .

The emergence of carbapenem-resistant represents a worldwide problem. To understand the carbapenem-resistance mechanisms and their spreading among strains, whole genome sequences were determined of two extensively drug-resistant strains that are endemic in Dutch hospitals. Strain Carb01 63 is of O-antigen serotype O12 and of sequence type ST111, whilst S04 90 is a serotype O11 strain of ST446.

Molecular Diagnosis of Urinary Tract Infections by Semi-Quantitative Detection of Uropathogens in a Routine Clinical Hospital Setting.

Background: The objective of our study was the development of a semi-quantitative real-time PCR to detect uropathogens. Two multiplex PCR reactions were designed to detect Escherichia coli, Klebsiella spp., Enterobacter spp.

The Widespread Presence of a Multidrug-Resistant Escherichia coli ST131 Clade among Community-Associated and Hospitalized Patients.

Background & Aims: The extent of entry of multidrug-resistant Escherichia coli from the community into the hospital and subsequent clonal spread amongst patients is unclear. To investigate the extent and direction of clonal spread of these bacteria within a large teaching hospital, we prospectively genotyped multidrug-resistant E. coli obtained from community- and hospital associated patient groups and compared the distribution of diverse genetic markers.

Within-Host and Population Transmission of blaOXA-48 in K. pneumoniae and E. coli.

During a large hospital outbreak of OXA-48 producing bacteria, most K. pneumoniaeOXA-48 isolates were phenotypically resistant to meropenem or imipenem, whereas most E. coliOXA-48 isolates were phenotypically susceptible to these antibiotics.

Laboratory Diagnosis of Pertussis.

The introduction of vaccination in the 1950s significantly reduced the morbidity and mortality of pertussis. However, since the 1990s, a resurgence of pertussis has been observed in vaccinated populations, and a number of causes have been proposed for this phenomenon, including improved diagnostics, increased awareness, waning immunity, and pathogen adaptation. The resurgence of pertussis highlights the importance of standardized, sensitive, and specific laboratory diagnoses, the lack of which is responsible for the large differences in pertussis notifications between countries.

Evaluation of different real time PCRs for the detection of Pneumocystis jirovecii DNA in formalin-fixed paraffin-embedded bronchoalveolar lavage samples.

The presence of Pneumocystis jirovecii in fresh clinical materials can be detected by PCR with high sensitivity and is thus preferred over microscopic methods. However, fresh materials are not always available, and on formalin-fixed paraffin-embedded materials, PCR may result in reduced detection rates. In this study the diagnostic sensitivity of P.

Screening rectal swabs for carbapenemase genes.

In an outbreak setting, we screened 16,296 samples from 3,644 patients by PCR for the presence of blaOXA-48, blaVIM, blaIMP, blaNDM, and blaKPC. The blaOXA-48 gene was found in samples from 43 patients infected with 9 different species of Enterobacteriaceae. Five patients had Pseudomonas aeruginosa isolates containing blaVIM.

Multi-centre evaluation of real-time multiplex PCR for detection of carbapenemase genes OXA-48, VIM, IMP, NDM and KPC.

Background: Resistance to carbapenem antibiotics is emerging worldwide among Enterobacteriaceae. To prevent hospital transmission due to unnoticed carriage of carbapenemase producing micro-organisms in newly admitted patients, or follow-up of patients in an outbreak setting, a molecular screening method was developed for detection of the most prevalent carbapenemase genes; blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC.

Methods: A real-time multiplex PCR assay was evaluated using a collection of 86 Gram negative isolates, including 62 carbapenemase producers.

Review of a major epidemic of methicillin-resistant Staphylococcus aureus: the costs of screening and consequences of outbreak management.

Background: A major outbreak of methicillin-resistant Staphylococcus aureus (MRSA) occurred in locations C and Z of our hospital and lasted for several years. It affected 1,230 patients and 153 personnel.

Methods: Outbreak management was installed according to the Dutch "search and destroy" policy.

Rapid HLA-B27 screening with real-time TaqMan PCR: a clinical validation in the Dutch population.

Background: Human leukocyte antigen B27 (HLA-B27) is strongly associated with ankylosing spondylitis. The B27 allele is present in 90% of patients with this disease, whereas it is present in only 9% of Caucasians. Molecular detection of HLA-B27 is traditionally based on allele specific amplification of exon 2 (Olerup method) or exon 3 (Dominguez method) by PCR, followed by gel analysis.

Detection of novel chromosome-SCCmec variants in Methicillin Resistant Staphylococcus aureus and their inclusion in PCR based screening.

Findings: To facilitate automation, a novel DNA extraction method for MRSA was adopted. The MRSA specific chromosome-SCCmec PCR was adapted, additional primers were added, and the performance was validated. From various laboratories in The Netherlands we received a total of 86 MRSA clinical isolates, that were negative in commercially available tests.

A real-time PCR assay with improved specificity for detection and discrimination of all clinically relevant Bordetella species by the presence and distribution of three Insertion Sequence elements.

Background: In Dutch laboratories molecular detection of B. pertussis and B. parapertussis is commonly based on insertion sequences IS481 and IS1001, respectively.

Molecular evidence for the ubiquitous presence of Legionella species in Dutch tap water installations.

Our aim was to investigate the occurrence and identity of Legionella spp. in Dutch tap water installations using culture, real-time PCR and sequence analysis. The PCR assays used were a 16S rRNA gene based PCR with both a Legionella species specific probe and a L.

Evaluation of real-time PCR for the early detection of Legionella pneumophila DNA in serum samples.

Legionella pneumonia can be difficult to diagnose. Existing laboratory tests all have shortcomings, especially in the ability to diagnose Legionnaires' disease (LD) at an early stage of the disease in a specimen that is readily obtainable. The aim of this study was to assess the performance of PCR as a rapid diagnostic method and to compare the results of different PCR assays of serum samples from patients with LD.

Bordetella holmesii DNA is not detected in nasopharyngeal swabs from Finnish and Dutch patients with suspected pertussis.

Bordetella holmesii is a Gram-negative bacterium first identified in 1995. It can cause pertussis-like symptoms in humans. B.

Detection and quantification of Legionella pneumophila DNA in serum: case reports and review of the literature.

Legionella pneumonia can be difficult to diagnose. Existing laboratory tests all have shortcomings, especially the ability to diagnose all Legionella spp. at an early stage.

Recognition of SCCmec types according to typing pattern determined by multienzyme multiplex PCR-amplified fragment length polymorphism analysis of methicillin-resistant Staphylococcus aureus.

Multienzyme multiplex PCR-amplified fragment length polymorphism (ME-AFLP) typing is a reliable and simple method for typing of bacterial species. In this study we analyzed two well-documented strain collections of Staphylococcus aureus and compared ME-AFLP typing results with results of various other typing methods. The discriminatory power of ME-AFLP was found comparable to pulsed-field gel electrophoresis, and typing results were highly concordant.

Comparison of three Legionella urinary antigen assays during an outbreak of legionellosis in Belgium.

During an outbreak of legionellosis in Belgium, urine samples of 32 legionellosis patients were tested with three Legionella urinary antigen assays: the Biotest enzyme immunoassay (EIA) kit, the Binax EIA kit and the Binax NOW Immunochromatographic Test kit. The three tests were concomitantly compared. The test sensitivities on the first urine samples were 65.

Outbreak of infection with a multiresistant Klebsiella pneumoniae strain associated with contaminated roll boards in operating rooms.

An outbreak with a multiresistant Klebsiella pneumoniae (MRKP) strain among seven patients admitted to the adult intensive care unit (ICU) of a regional teaching hospital in The Netherlands was investigated. Epidemiologic investigations revealed a short delay between an operation and the acquisition of the MRKP strain. A case-control study comprising 7 cases and 14 controls was conducted to identify the risk factors associated with the acquisition of the MRKP strain.

Effect of clarithromycin treatment on Chlamydia pneumoniae in vascular tissue of patients with coronary artery disease: a randomized, double-blind, placebo-controlled trial.

Several small clinical trials have indicated that antibiotic treatment of Chlamydia pneumoniae infection is associated with a better outcome in patients with coronary artery disease (CAD). It has not been demonstrated whether antibiotic treatment eradicates C. pneumoniae from vascular tissue.

Is the perceived association between Chlamydia pneumoniae and vascular diseases biased by methodology?

Inter- and intralaboratory inconsistencies in detection rates of Chlamydia pneumoniae in vascular specimens have been demonstrated. In this study, 66 vascular tissue specimens from 66 patients with vascular disease were tested by three PCR assays: a 16S PCR-based reverse line blot (RLB) assay, a single-step PCR, and a nested PCR. Also, we explored the impacts of different DNA polymerase enzymes on the results based on gel electrophoresis and hybridization.

Diagnosis of atypical pathogens in patients hospitalized with community-acquired respiratory infection.

The object of our study was to determine the proportion of atypical respiratory pathogens among patients hospitalized with a community-acquired respiratory infection. From September 1997 to May 1999, 159 patients (57% male, median age 55, range 1-88 y) admitted to 3 regional hospitals for a community acquired respiratory infection, were enrolled in the study. Microbiological diagnosis for the atypical pathogens Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella pneumophila was performed with PCR on a throat swab, sputum and/or broncho alveolar lavage (BAL).

Polymerase chain reaction detection of Neisseria meningitidis in the intraocular fluid of a patient with endogenous endophthalmitis but without associated meningitis.

Purpose: To report a patient with Neisseria meningitidis endophthalmitis without associated meningitis with full visual recovery, with early detection of the microorganism using polymerase chain reaction (PCR) analysis.

Design: Retrospective, observational case report.

Participants: One patient with endogenous endophthalmitis.

Evaluation of real-time PCR for detection of and discrimination between Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii for clinical diagnosis.

PCR is increasingly being used as a diagnostic test for the detection of Bordetella pertussis and Bordetella parapertussis DNA, as it has improved sensitivity and specificity in comparison to conventional techniques. The assay described here uses the two insertion sequences IS481 and IS1001 for B. pertussis and B.