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Within the last few years, methicillin-resistant Staphylococcus aureus (MRSA) strains encoding Panton-Valentine leukocidin (PVL) have emerged and spread worldwide. This epidemic can be attributed to a small number of distinct clones. The present study used a novel assay, based on multiplex linear DNA amplification and subsequent microarray hybridisation, to simultaneously detect all relevant exotoxins, antimicrobial resistance determinants and the allelic variants of agr. The genes of the staphylococcal exotoxin-like (set) locus were also included for typing purposes. This assay, together with multilocus sequence typing (MLST) and spa typing, was applied to 56 clinical isolates and reference strains representing all major pandemic PVL-MRSA lineages, as well as to phylogenetically-related strains and putative ancestors. Array hybridisation results allowed the assignment of isolates to clonal groups, which were in accordance with MLST and spa typing data. Ten distinct clonal groups of PVL-MRSA (ST1, ST5, ST8, ST22, ST30, ST59/359, ST80/583, ST88, ST93 and ST152), including 12 MLST types, were identified and analysed with regard to resistance determinants and genes coding for exotoxins. The array hybridisation data confirmed that pandemic PVL-positive strains originate from very diverse genetic backgrounds, and provided insights into the evolution of some lineages. The DNA microarray technique provides a valuable epidemiological tool for the detailed characterisation of clinical isolates and comparison of strains at a global level.

We developed a standardized DNA sequence-based approach for the accurate and timely identification of medically important fungi by sequencing polymerase chain reaction (PCR) products with a rapid automated capillary electrophoresis system. A simple DNA extraction method and PCR amplification using universal fungal primers was used to amplify ribosomal DNA from a range of clinical isolates and reference strains. The entire internal transcribed spacer (ITS) 1-5.8S-ITS2 ribosomal DNA region was sequenced using automated dye termination sequencing for 89 clinical isolates. These had previously been identified by traditional methods and included 12 ascomycetous yeast species, three basidiomycetous yeast species, eight dermatophyte species and two thermally dimorphic fungi, Scedosporium prolificans and S. apiospermum. Furthermore, 21 reference strains representing 19 different Candida species, Geotrichum candidum and Malassezia furfur were also sequenced as part of this study and were used either as standards for sequence-based comparisons, or as assay controls. Sequence-based identification was compared to traditional identification in a blinded manner. Of the clinical isolates tested, 88/89 had DNA sequences that were highly homologous to those of reference strains accessioned in GenBank, and 87/89 gave a sequence-based identification result that correlated with the traditional identification. In contrast to relatively slow conventional methods of identification, a sequence-based identification from a pure culture can be obtained within 24 h of a DNA extraction carried out after a minimal period of culture growth. We conclude that this approach is rapid, and may be a more accurate cost-effective alternative than most phenotypic methods for identification of many medically important fungi frequently encountered in a routine diagnostic microbiology laboratory.

Objective:To demonstrate that nosocomial transmission of vancomycin-resistant enterococci (VRE) can be terminated and endemicity prevented despite widespread dissemination of an epidemic strain in a large tertiary-care referral hospital.Interventions:Two months after the index case was detected in the intensive care unit, 68 patients became either infected or colonized with an epidemic strain of vanB vancomycin-resistantEnterococcus faeciumdespite standard infection control procedures. The following additional interventions were then introduced to control the outbreak: (1) formation of a VRE executive group; (2) rapid laboratory identification (30 to 48 hours) using culture and polymerase chain reaction detection ofvanA andvanBresistance genes; (3) mass screening of all hospitalized patients with isolation of carriers and cohorting of contacts; (4) environmental screening and increased cleaning; (5) electronic flagging of medical records of contacts; and (6) antibiotic restrictions (third-generation cephalosporins and vancomycin).Results:A total of 19,658 patient and 24,396 environmental swabs were processed between July and December 2001. One hundred sixty-nine patients in 23 wards were colonized with a single strain of vanB vancomycin-resistantE. faecium.Introducing additional control measures rapidly brought the outbreak under control. Hospital-wide screening found 39 previously unidentified colonized patients, with only 7 more nonsegregat-ed patients being detected in the next 2 months. The outbreak was terminated within 3 months at a cost of $2.7 million (Australian dollars).Conclusion:Despite widespread dissemination of VRE in a large acute care facility, eradication was achievable by a well-resourced, coordinated, multifaceted approach and was in accordance with good clinical governance.

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A real-time PCR assay previously developed for use on the Roche LightCycler platform was investigated as an alternative to culture for the direct detection of vancomycin-resistant enterococci (VRE) in clinical specimens. PCR primers and fluorescence resonance energy transfer hybridization probes specific for the vanA and vanB genes were combined in a multiplex real-time PCR assay performed directly with fecal material obtained by rectal swabbing and with enrichment broth samples. DNA was prepared from the rectal swabs and enrichment broths with a commercially available DNA preparation column designed specifically for use with fecal specimens. One hundred eighty duplicate rectal swabs were obtained from 42 patients who were previously found to be positive for VRE and who were being monitored for carriage of VRE. Direct and enrichment broth cultures were performed with one swab, while PCR was performed with the other swab as well as any corresponding presumptive positive enrichment broth. In total, 100 specimens from 30 patients remained positive for VRE by at least one method. The multiplex real-time PCR was positive for 88 enrichment broths of rectal swabs from 27 patients but for only 45 rectal swabs from 15 patients. Direct culture was positive for VRE for only 43 specimens from 11 patients, while enrichment broth culture was positive for VRE for 75 specimens from 22 patients. Inhibition studies for the multiplex real-time PCR assay, performed by spiking the DNA extracts from 50 negative rectal swabs and the corresponding enrichment broths with between 1 and 10 CFU of a VanB Enterococcus faecium strain, detected inhibition rates of 55.1 and 10%, respectively. PCR performed directly with enrichment broths was found to be significantly more sensitive than enrichment broth culture (P < 0.025). Negative samples were identified significantly earlier by PCR than by culture alone.

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