The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood. INTRODUCTIONBlood culture remains the accepted 'gold standard' method for diagnosis of candidiasis and can be highly sensitive. However, in some settings, culture can fail to detect Candida spp. in more than 50 % of patients with chronic disseminated candidiasis MoreiraOliveira et al., 2005). Molecular methods, particularly realtime PCR, are increasingly used alongside conventional microbiological techniques for the diagnosis of systemic candidiasis (Klingspor & Jalal, 2006; Kasai et al., 2006; Löffler et al., 2000;Schabereiter-Gurtner et al., 2007;Bretagne & Costa., 2005;White et al., 2006;Maaroufi et al., 2003; Moreira-Oliveira et al., 2005). However, two major limitations of PCR are the difficulty associated with breaking yeast cell walls to release Candida spp. DNA suitable for amplification (Maaroufi et al., 2004; Löffler et al., 1997) and limited sensitivity when the assays are adapted for testing clinical specimens, such as blood samples, in which the amounts of fungal DNA may be very low (White et al., 2003; Löffler et al., 2000;Ahmad et al., 2002;Challier et al., 2004). As a consequence, these tests are not yet recognized as part of the consensus diagnostic criteria, in contrast to some antigenaemia detection kits (Ascioglu et al., 2002).The yeast cell is notoriously difficult to lyse due to a highly complex cell wall structure that provides rigidity (Müller et al., 1998). Moreover, it has been suggested that the fungal load in blood from ...
Caprbapenemase-producing Gram-negative micro-organisms are emerging as a major clinical problem. The infections caused by these highly resistant and hospital-adapted pathogens may become untreatable using existing antibiotics. Over a three year period, six patients at a large UK tertiary-referral hospital were colonised or infected with carbapenem-resistant Enterobacter cloacae carrying the blaNDM-1 metallo-β-lactamase gene. Environmental isolates were also obtained from a clinical wash-hand basin and taps. The isolates had very similar pulsed-field gel electrophoresis profiles, suggesting they were related, although only four of the cases had epidemiological links. Whole genome sequencing showed the isolates had the same genomic background (sequence type ST114). Genes encoding seven different extended-spectrum and inhibitor-resistant β-lactamase and carbapenemase enzymes (blaNDM-1; blaCTX-M-15; blaACT-16; blaVEB-1; blaTEM-1; blaOXA-1 and blaOXA-10) were present, in addition to multiple genes and mutations conferring resistance to aminoglycosides, quinolones, trimethoprim, tetracycline, sulphonamide, chloramphenicol, rifampicin and fosfomycin. Phenotypic testing indicated sensitivity only to colistin and tigecycline. Genome-wide single nucleotide polymorphism analysis showed the four linked isolates were closely related, and differed from the unlinked isolates by 16–24 SNPs. Moreover, resistance encoding plasmids had been lost in the two unlinked isolates. This suggested these isolates, although sharing a recent common ancestor, had evolved in different environments. Whole genome sequencing allowed resolution of very closely related E. cloacae strains, and confirmed the outbreak did not extend beyond the linked patients. Sequencing also confirmed the same highly resistant E. cloacae strain had persisted within a clinical unit for over two years, despite rigorous efforts to eradicate it.
Background: We describe an outbreak of Clostridium difficile infection (CDI) with ribotype 053, a possible hypervirulent strain that causes outbreaks, in a neurosurgical unit. Outbreak investigation:The outbreak was investigated by creating a timeline of all toxin positive patients with root cause analysis, supplemented with ribotyping results, hand hygiene and environmental audits. There were five cases of CDI, three caused by ribotype 053 indicating transmission. Infection prevention measures:These included a short period of ward closure to allow enhanced cleaning, including use of vaporised hydrogen peroxide, isolation of infected patients, reinforcement of hand hygiene, education of all staff on C. difficile, reduction of shared bay occupancy from six to four, and addressing staffing levels. Discussion:The patients with ribotype 053 all had long inpatient stays and had required several courses of broadspectrum antibiotics for aspiration pneumonia. They also required enteral feeding, which can cause diarrhoea, and during long inpatient stays they had multiple toxin negative faecal samples making clinical diagnosis of CDI difficult. Hence they were not isolated promptly, leading to transmission. This is the first reported outbreak of C. difficile ribotype 053 in the UK and highlights the unique aspects of an outbreak in neurosurgical patients.
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