SummaryBackground Gaps in the diagnostic capacity and heterogeneity of national surveillance and reporting standards in Europe make it diffi cult to contain carbapenemase-producing Enterobacteriaceae. We report the development of a consistent sampling framework and the results of the fi rst structured survey on the occurrence of carbapenemaseproducing Klebsiella pneumoniae and Escherichia coli in European hospitals.

This study demonstrates that multimodal prevention strategies aiming at improving CVC insertion practice and HH reduce CRBSI in diverse European ICUs. Compliance explained CRBSI reduction and future quality improvement studies should encourage measuring process indicators.

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A 30 month prospective study of Acinetobacter species encountered in the Central Pathology Laboratory of St James's Hospital, Dublin, Ireland, was conducted to investigate the prevalence and molecular epidemiology of carbapenem resistance in such isolates. Acinetobacter genomic species 3 (AG3) was found to be the predominant Acinetobacter species (45/114, 39 %) in our institution. A total of 11 % of all Acinetobacter species (12/114) and 22 % of AG3 isolates (10/ 45) were carbapenem resistant. Carbapenem resistance was mediated by Ambler class D blactamase OXA-23 in all 12 isolates, with insertion sequence ISAba1 found upstream of bla OXA-23 . ISAba1 was also found upstream of bla ADC-25 , which encodes the enzyme AmpC, in an Acinetobacter baumannii isolate, and upstream of the aminoglycoside-acetyltransferaseencoding gene aacC2 in three AG3 isolates. Inter-species plasmidic transfer was most likely involved in the emergence and spread of bla OXA-23 among the Acinetobacter isolates within our institution. The emergence of carbapenem resistance was associated not only with prior carbapenem use but also with the use of other antimicrobial agents, most notably b-lactam/blactamase-inhibitor combinations. The study demonstrated the emerging trend of carbapenem resistance in the wider context of the Acinetobacter genus, and reiterated the paramount importance of the prudent use of antimicrobial agents, stringent infection control measures and resistance surveillance of pathogens.

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BackgroundStreptococcus pneumoniae is an important cause of microbial disease in humans. The introduction of multivalent vaccines has coincided with a dramatic decrease in the number of pneumococcal-related deaths. In spite of this, at a global level, pneumococcal infection remains an important cause of death among children under 5 years of age and in adults 65 years of age or older. In order to properly manage patients and control the spread of infection, a rapid and highly sensitive diagnostic method is needed for routine implementation, especially in resource-limited regions where pneumococcal disease is most prevalent.MethodsUsing the gene encoding leader peptidase A as a molecular diagnostics target, a real-time RPA assay was designed and optimised for the detection of S. pneumoniae in whole blood. The performance of the assay was compared to real-time PCR in terms of its analytical limit of detection and specificity. The inhibitory effect of human genomic DNA on amplification was investigated. The potential clinical utility of the assay was investigated using a small number of clinical samples.ResultsThe RPA assay has a limit of detection equivalent to PCR (4.0 and 5.1 genome equivalents per reaction, respectively) and was capable of detecting the equivalent of <1 colony forming unit of S. pneumoniae when spiked into human whole blood. The RPA assay was 100 % inclusive (38/38 laboratory reference strains and 19/19 invasive clinical isolates) and 100 % exclusive; differentiating strains of S. pneumoniae species from other viridans group streptococci, including S. pseudopneumoniae. When applied to the analysis of a small number (n = 11) of clinical samples (blood culture positive for S. pneumoniae), the RPA assay was demonstrated to be both rapid and sensitive.ConclusionsThe RPA assay developed in this work is shown to be as sensitive and as specific as PCR. In terms of reaction kinetics, the RPA assay is shown to exceed those of the PCR, with the RPA running to completion in 20 minutes and capable generating a positive signal in as little as 6 minutes. This work represents a potentially suitable assay for application in point-of-care settings.

Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae, N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology.

There were two parts to this study. Part 1 evaluated the epidemiology of Candida bloodstream isolates within the Southern Health Board (SHB) of Ireland from 1992 to 2003 by retrospective surveillance of all such isolates of patients reported from SHB hospitals to our laboratory database during that period. Part 2 reviewed candidaemia cases occurring in Cork University Hospital (CUH) from 1999 to 2003 using surveillance of all positive blood culture isolates in CUH microbiology laboratory during the 5-year period. In part 1, 250 Candida bloodstream isolates were reported in the SHB over 12 years. There was a pattern of decreasing percentage of C. albicans with time. Whereas in part 2, 63 cases of candidaemia were identified in CUH from 1999 to 2003. Candida albicans constituted 50% of all isolates, while C. parapsilosis and C. glabrata accounted for 21.2% and 18.2% respectively. Average annual incidence rate was 0.48 episodes/1000 admissions and 0.70 episodes/10 000 patient-days. Vascular catheters were the commonest source of candidaemia (61.9%) followed by the urinary tract (12.7%). Risk factors included exposure to multiple antibiotics (75%); central vascular catheterization (73%); multiple colonization sites (71%); severe gastrointestinal (GI) dysfunction (54%) and acute renal failure (43%). Crude 7-day and 30-day mortality rates were 20.6% and 39.7% respectively. Logistic regression multivariate analysis identified the following to be independent predictors for mortality: age > or =65 years [odds ratio (OR) 7.2, P = 0.013]; severe GI dysfunction (OR 10.6, P = 0.01); acute renal failure (OR 7.6, P = 0.022); recent/concurrent bacteraemia (OR 5.2, P = 0.042); endotracheal intubation (OR 7.7, P = 0.014); while major surgery was associated with a better prognosis (OR 0.05, P = 0.002). Appropriate antifungal treatment was also found to be associated with survival (Fisher's exact test, P < 0.001). The epidemiology of Candida bloodstream isolates within our health board had changed over the years. Incidence and mortality of candidaemia were relatively high in our hospital. Dysfunction of major organ systems and recent bacteraemia were found to predict mortality.