Repeated introduction and spread of the MRSA clone t304/ST6 in northern Europe.

OBJECTIVES: During the last decades several methicillin-resistant Staphylococcus aureus (MRSA) clones with the capability of global spread have emerged in the community. Here, we have investigated a large collection of clinical isolates belonging to MRSA clone t304/ST6, which has emerged in many European countries over the last years, in order to retrace its phylogeny and its spread. METHODS: We characterized 466 ST6 isolates from Denmark (n = 354), France (n = 10), Norway (n = 24), Sweden (n = 27) and the UK (n = 51). All had spa-type t304 (n = 454) or t304-related spa-types (n = 12) and whole genome sequencing (WGS) was carried out on Illumina Miseq or Hiseq with 100-300 bp reads. cgMLST was performed using Ridom SeqSphere. RESULTS: A minimum spanning tree (MST) of all 466 isolates showed one large cluster including 182 isolates collected only from Denmark and related to a long-term neonatal outbreak in Copenhagen. This cluster contrasted with numerous small clusters, including the remaining Danish isolates and isolates from the other countries that interspersed throughout the tree. Most isolates were Panton-Valentine leukocidin (PVL) negative (95%) and harboured SCCmec IVa. One genome was closed using Oxford Nanopore technology and Illumina MiSeq. It contained a plasmid of 19.769 bp including the blaZ gene. A similar plasmid was found in 78% of all isolates. DISCUSSION: t304/ST6 is a successful emerging clone and the fact that isolates from five countries are interspersed throughout the MST indicates a common origin. This clone is commonly described in the Middle East and its emergence in Europe coincides with influx of refugees from the Syrian Civil War.

Commercial bacterial and fungal broad-range PCR (Micro-Dx™) used on culture-negative specimens from normally sterile sites: diagnostic value and implications for antimicrobial treatment.

The aim of this study was to evaluate the clinical value of partial 16S/18S rRNA gene sequencing with the commercial kit Micro-Dx™ used with the SelectNA™plus instrument on culture-negative samples. A retrospective study of microbiological and clinical data from a 2.5-year period was performed. Assessment of the clinical relevance of the 16S/18S rRNA gene sequencing results was based on evaluation of the results in the clinical context and changes in antimicrobial therapy. Included were 529 samples from 223 patients, representing 251 episodes. In 191 samples (36.1%), bacterial/fungal DNA was detected. Positive results were judged clinically relevant in 79 (31.5%) episodes. Antimicrobial treatment was adjusted according to the 16S/18S rRNA gene sequence analysis result in 42 (16.7%) episodes. The results from 16S/18S rRNA gene sequence analysis were highly clinically relevant. These findings support the use of this analysis in a routine setting.

Typing of vancomycin-resistant enterococci with MALDI-TOF mass spectrometry in a nosocomial outbreak setting.

OBJECTIVES: To investigate the usefulness of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) typing as a first-line epidemiological tool in a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VREfm). METHODS: Fifty-five VREfm isolates, previously characterized by whole-genome sequencing (WGS), were included and analysed by MALDI-TOF MS. To take peak reproducibility into account, ethanol/formic acid extraction and other steps of the protocol were conducted in triplicate. Twenty-seven spectra were generated per isolate, and spectra were visually inspected to determine discriminatory peaks. The presence or absence of these was recorded in a peak scheme. RESULTS: Nine discriminatory peaks were identified. A characteristic pattern of these could distinguish between the three major WGS groups: WGS I, WGS II and WGS III. Only one of 38 isolates belonging to WGS I, WGS II or WGS III was misclassified. However, ten of the 17 isolates not belonging to WGS I, II or III displayed peak patterns indistinguishable from those of the outbreak strain. CONCLUSIONS: Using visual inspection of spectra, MALDI-TOF MS typing proved to be useful in differentiating three VREfm outbreak clones from each other. However, as non-outbreak isolates could not be reliably differentiated from outbreak clones, the practical value of this typing method for VREfm outbreak management was limited in our setting.

Screening for vancomycin-resistant enterococci with Xpert® vanA/vanB: diagnostic accuracy and impact on infection control decision making.

Vancomycin-resistant enterococci (VRE) are increasingly important nosocomial pathogens and screening for colonization status is a mainstay in infection control. We implemented PCR-based screening during vanA-positive Enterococcus faecium outbreaks in four university hospitals in Copenhagen, Denmark. Xpert®vanA/vanB was performed directly on rectal swabs and the vanA PCR result was used to guide infection control measures. Concurrently, all samples were selectively cultured including an overnight enrichment step. Diagnostic accuracy was calculated as well as turnaround time and the impact of the earlier available PCR results on infection control decision making. In all, 1110 samples were analysed. The vanA PCR positivity rate was 13.8% and culture positivity rate was 15.2%. The diagnostic accuracy of the vanA part of the assay was high with a sensitivity of 87.1%, a specificity of 99.7%, and positive and negative predictive values of 98.0% and 97.7%, respectively. The vanB PCR had a considerably lower specificity of 77.6% and a positive predictive value of 0.4%. In 1067 (96.1%) samples, PCR results were reported within 1 day, whereas median culture turnaround time was 3 days. The saving of time to available results corresponded to 141 saved isolation days and 292 saved transmission risk days. False-negative or false-positive PCR results led to six additional transmission risk days and 13 additional isolation days, respectively. The vanA PCR had high diagnostic accuracy and the prompt availability of results gave a considerable benefit for infection control decision making.

Decreasing candidaemia rate in abdominal surgery patients after introduction of fluconazole prophylaxis*.

Although abdominal surgery is an established risk factor for invasive candidiasis, the precise role of antifungal prophylaxis in these patients is not agreed upon. In 2007, fluconazole was added to the prophylactic antibiotic treatment for patients with gastrointestinal tract perforations or reoperation after colorectal surgery in two university hospitals in Copenhagen. Changes in candidaemia rates associated with this intervention were examined and potential interfering factors evaluated. Rates and clinical characteristics of candidaemias and other blood stream infections (BSIs) in abdominal surgery patients were compared before (1 January 2006 to 30 June 2007) and after the intervention (1 January 2008 to 30 June 2009). The departments' activity was assessed by number of bed-days, admissions and surgical procedures, and the consumption of antifungals was analysed. The candidaemia rate decreased from 1.5/1000 admissions in the pre-intervention to 0.3/1000 admissions in the post-intervention period (p 0.002). Numbers of BSIs and bed-days remained stable, and numbers of admissions and surgical procedures performed increased during the study period. Fluconazole consumption in the two abdominal surgery departments increased from 4.6 to 12.2 defined daily doses per 100 bed-days (p <0.001), and 3.2 to 5.0 (p 0.01), respectively, but remained unchanged in the intensive care units. We could not detect any increase in fluconazole-resistant strains (14/29 pre- and 2/7 post-intervention, p 0.43). The introduction of fluconazole prophylaxis was followed by a significantly decreased candidaemia rate. However, the observational study design does not allow conclusions regarding causality. No increase in resistance was detected, but follow-up was short and continuing surveillance is needed.